GPR 17 agonists and screening assay

ABSTRACT

The present invention is related to a method of determining a test compound&#39;s ability to modify the biological activity of a GPR17. Said method comprises, among others, the step of contacting the test compound with a GPR17, or a functional GPR17 fragment in the presence of a suitable amount of a GPR17 agonist of formula I.

This application is a divisional application of U.S. patent applicationSer. No. 13/407,512, filed Feb. 28, 2012, which claims the benefit ofEuropean Patent Application 11180467.0 filed Sep. 7, 2011.

BACKGROUND

G-protein coupled receptors (GPCRs) constitute the largest family ofmembrane receptors in the cell. They transduce extracellular signals tointracellular effector systems and are involved in a large variety ofphysiological phenomena, therefore representing the most common targetof pharmaceutical drugs although only a small percentage of GPCRs aretargeted by current therapies.

GPCRs respond to a wide range of ligands. Due to the progress of humangenome sequencing, for about 25% out of the more than 400 GPCRs (notincluding the olfactory GPCRs) that have been identified, a definedphysiologically relevant ligand is still lacking. These receptors areknown as “orphan GPCRs”. “Deorphanization” and identification of theirin vivo roles is expected to clarify novel regulatory mechanisms and,therefore, to disclose novel drug targets. Whether GPR17 is such anorphan receptor is still a matter of debate. Phylogenetically, GPR17 ismost closely related to the nucleotide P2Y receptors and thecysteinylleukotriene (CysLT1, CysLT2) receptors, with an amino acidsequence identity of between about 30 and about 35%, respectively.

Multiple-tissue Northern blot and RT-PCR analyses indicate a predominantexpression of GPR17 in the central nervous system (CNS) (Ciana et al.,2006, EMBO J 25(19): 4615; Blasius et al., 1998, J Neurochem 70(4):1357) and additionally in heart and kidney, i.e. organs typicallyundergoing ischemic damage. Two GPR17 isoforms have been identifieddiffering only by the length of their N-terminus. The short GPR17isoform encodes a 339 amino acid-residue protein with typical rhodopsintype-seven transmembrane motifs. The long isoform encodes a receptorwith a 28 amino acid longer N-terminus (Blasius et al., 1998). GPR17 ishighly conserved among vertebrate species (˜90% identity of amino acidsequence to both mouse and rat orthologs), which may constitute anadvantageous feature for development of small molecule ligands andanimal models in a drug discovery context.

In the original deorphaning report, GPR17 was identified as dualreceptor for uracil nucleotides and cysteinyl-leukotrienes (cysLTs) LTC4and LTD4, respectively based on ³⁵SGTPγS binding and cAMP inhibitionassays as well as single cell calcium imaging (Ciana et al., 2006).Evidence for GPR17 functionality was provided in different cellularbackgrounds such as 1321N1, COS7, CHO, and HEK293 cells (Ciana et al.,2006). Subsequently, an independent study confirmed activation of GPR17by uracil nucleotides but failed to recapitulate activation by CysLTs(Benned-Jensen, 2010, Br J Pharmacol, 159(5): 1092). Yet another veryrecent report (Maekawa et al., 2009) suggested lack of GPR17responsiveness to both uracil nucleotides and CysLTs across threedifferent cellular backgrounds stably expressing GPR17 (1321N1, CHO,HEK293 cells). Instead a novel regulatory role for GPR17 was proposed:GPR17—upon coexpression with CysLT1—rendered CysLT1 unresponsive to itsendogenous lipid mediators LTC4 and LTD4. Clearly, additional in vitroinvestigations are required to probe GPR17 pharmacology and function inmore depth.

Drugs modulating the GPR 17 activity may have neuroprotective,anti-inflammatory and anti-ischemic effects and may thus be useful forthe treatment of cerebral, cardiac and renal ischemia, and stroke (WO2006/045476). WO 2005/103291 disclosed analgetic effects of a GPR 17agonist and proposed the use of GPR 17 agonists for treating neuropathicpain. Moreover, evidence is accumulating that GPR 17 is involved inmyelination processes and that GPR 17 antagonists can be valuable drugsfor the treatment or alleviation of myelination disorders such asmultiple sclerosis or spinal cord injury (Chen et al, Natureneuroscience 2009, 12(11):1398-406; Ceruti et al; Brain: a journal ofneurology 2009 132(Pt 8):2206-18). The identification of potent andselective GPR 17 modulators could thus be of significant relevance inthe treatment of these serious diseases.

Identification of an activating ligand is a prerequisite to search forcompounds that modulate GPR17 activity. Although activation by uracilnucleotides and cysteinyl-leukotrienes of GPR17 has been reported (Cianaet al., 2006; Pugliese et al., 2009, Am J Physiol Cell Physiol 297:C1028), these endogenous signalling molecules do not display functionalactivity in different cell lines (1321N1, CHO, HEK) engineered to stablyexpress the short isoform of GPR17 in our laboratory. In agreement withour observations, inactivity of these ligand classes has also beenobserved in a recent study (Maekawa et al., 2009, PNAS, US, 106(28):11685). Another independent laboratory was also not able to confirmcysteinyl-leukotrienes as GPR17 agonists (Benned-Jensen, 2010), whiledepending on the GPR 17 isoform tested some functional activity ofuracil nucleotides was seen, although only at low, or high micromolarconcentrations, respectively.

WO 2005/103291 suggested the endogenous molecules 5 amino levulinic acid(5-ALA) and porphobilinogen (PBG) as activating ligands for GPR17, and ascreening assay using these GPR 17 agonists. However, the reportedaffinity of 5-ALA and PBG is quite low and the amounts needed in theassays are significant, namely in the three digit micromolar range for5-ALA or even in the mM range for PBG, which make both compounds notwell suited for use in routine screening assays or even high throughputscreenings. Moreover, PBG is a chemical unstable, reactive compoundwhich rapidly decomposes after exposure to air and light, making itimpractical to handle on a routine basis.

Accordingly, a need exists for the identification of improved GPR 17agonists, which can be used as an easy and cheep but robust and reliabletool for the identification of GPR17 antagonists in various experimentalsettings.

3-(2-Carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid and someanalogs had been previously described as allosteric inhibitors offructose-1,6-biphosphatase (Wright et al, MBCL 2003, 13, 2055), and asantagonists of the NMDA receptor associated glycine binding site(Salituro, J Med Chem, 1992, 35, 1791; U.S. Pat. No. 4,960,786).However, these compounds have not yet been described as GPR17modulators.

SUMMARY OF THE INVENTION

We have now detected that3-(2-carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid (herein alsoreferred to as RA-II-150) and other compounds of formula I, infra, areagonists for GPR17. In contrast to cysteinylleukotrienes and uracilnucleotides, we have found that RA-II-150 and other compounds of formulaI and their salts activate GPR17 irrespective of the cellularbackground, i.e. they activate the receptor in a number of differentcell lines such as 1321N1 astrocytoma cells, human embryonic kidneyHEK293 cells, and Chinese hamster ovary (CHO) cells.

Table 1 and FIG. 1 disclose concentration-effect curves for themobilization of intracellular Ca²⁺ in human GPR17 (hGPR17) transfected1321 and CHO cells, respectively, by RA-II-150 and other compounds offormula I. None of these identified GPR17 agonists display activity innative cells that lack GPR17.

Table 1.

Potencies and efficacies of RA-II-150 and other compounds of formula Iin the 1321N1-GPR17 and CHO-GPR17 cell systems were determined from thecalcium mobilization assays described in Example 2, below. Data inrecombinant 1321N1 and CHO cells are normalised to the response of 30 μMand 0.3 μM RA-II-150 (column 3), respectively. Unspecific effects ofactive compounds are tested on native 1321N1 or CHO-K1 cells. Here, dataare normalised to the response of 100 μM carbachol or 100 μM ATP (column4) (n=3−4).

TABLE 1 % of response of % response of Chemical structure and pEC₅₀ 30μM compound 30 μM compound internal designation of (± SEM) (± SEM) (±SEM) selected compounds of 1321N1-GPR17/ 1321N1-GPR17/ Native 1321N1cells/ formula I CHO-GPR17 CHO-GPR17 cells CHO-K1 cells

6.09 (0.06)/ 8.20 (0.08) 100 (0)/100 (0) (0.3 μM compound) 0 (3)/0 (1)(0.1 μM compound)

5.28 (0.08)/ 6.73 (0.12) 80 (6)/85 (5) (1 μM compound) 1 (1)/1 (1) (1 μMcompound)

4.86 (0.10)/ 6.47 (0.05) 76 (6)/68 (2) (1 μM compound) 1 (1)/1 (1) (1 μMcompound)

5.43 (0.14)/ 7.69 (0.10) 84 (6)/94 (7) (1 μM compound) 4 (3)/1 (2) (0.3μM compound)

5.13 (0.13)/ 6.79 (0.10) 77 (2)/97 (5) (3 μM compound) 2 (1)/1 (2) (3 μMcompound)

6.71 (0.01)/ 7.87 (0.16) 106 (1) (10 μM compound)/89 (2) (0.3 μMcompound) 0 (1)/0 (0) (0.3 μM compound)

5.50

6.15

5.32

5.52

6.14

The following GPR17 agonists which are also structures of formula I alsoform part of the disclosure:

TABLE 2 Structure Name

3-(2-carboxyethyl)-6,7-dichloro-1H-indole-2-carboxylic acid(3-(2-Carboxy-6,7-dichloroindol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-chloro-7-fluoro-1H-indole-2- carboxylic acid(3-(2-Carboxy-6-chloro-7-fluoroindol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-bromo-2-carboxy-7-fluoro-1H- indole-2-carboxylicacid (3-(6-Bromo-2-carboxy-7-fluoroindol-3-yl)propionic acid)

3-(2-carboxyethyl)-4,6-dimethoxy-1H-indole-2- carboxylic acid(3-(2-Carboxy-4,6-dimethoxyindol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-phenoxy1H-indole-2-carboxylic acid,(3-(2-Carboxy-6-phenoxyindol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-benzyl-2-carboxy-1H-indole-2- carboxylic acid(3-(6-Benzyl-2-carboxyindol-3-yl)propionic acid)

3-(2-carboxyethyl)-4,6-dihydroxy-1H-indole-2- carboxylic acid(3-(2-Carboxy-4,6-dihydroxyindol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-(4-fluorophenyl)-1H-indol-2- carboxylic acid,(3-[2-Carboxy-6-(4-fluorophenyl)indol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-furanyl-1H-indole-2-carboxylic acid,(3-(2-Carboxy-6-furanylindol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-thienyl-1H-indole-2-carboxylic acid,(3-(2-Carboxy-6-thienylindol-3-yl)propionic acid)

3-(2-carboxyethyl)-7-fluoro-6-phenyl-1H-indole-2- carboxylic acid(3-(2-Carboxy-7-fluoro-6-phenylindol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-(4-fluorophenyl)-7-fluoro-1H- indol-2-carboxylicacid, (3-(2-Carboxy-6-(4-fluorophenyl)-7-fluoroindol-3- yl]propionicacid)

3-(2-carboxyethyl)-6-furanyl-7-fluoro-1H-indole-2- carboxylic acid(3-(2-Carboxy-6-furanyl-7-fluoroindol-3-yl)propionic acid)

3-(2-carboxyethyl)-6-thienyl-7-fluoro-1H-indole-2- carboxylic acid,(3-(2-Carboxy-6-thienyl-7-fluoroindol-3-yl)propionic acid)

A comparison of a representative GPR17 agonist of the present invention,RA-II-150, with the putative GPR 17 agonists 5-ALA and PBG (see WO2005/103291) revealed a higher affinity and selectivity of the presentlydisclosed GPR 17 agonists. While the inventors of the presentapplication were able to confirm the activation of GPR17 by 5-ALA, allattempts to activate GPR 17 with porphobilinogen failed. Also, aGPR17-independent inhibition of forskolin stimulated adenylylcyclaseactivity in GPR17 CHO cells was recognized after 5-ALA addition, thusquestioning the specificity of GPR17 activation by 5-ALA. Irrespectiveof the quality of 5ALA to serve as a selective stimulus for GPR17, it isconsiderably less potent (10.000 fold) as compared with the presentlydisclosed small molecule agonist RA-II-150 and its structural analogs(FIG. 2).

In summary, we identified small molecules with the ability to activateGPR17 with high specificity and potency and significant superiority overpublished GPR17 agonists. The GPR17 agonists of the present inventioncan be used for establishing functional GPR17 assays to search forinhibitors of said receptor to treat/prevent neurodegenerative diseasessuch as spinal cord injury, multiple sclerosis, cerebral, cardiac andrenal ischemia, and preferably multiple sclerosis, ischemic damage andstroke.

Accordingly, one aspect of the present invention relates to smallmolecule GPR17 agonists, as chemically further defined below, andmethods for using the agonists in the identification of and screeningfor GPR17 modulators, in particular GPR 17 antagonists.

One aspect of the present invention relates to screening assayscomprising the GPR17 agonists of the present invention and cells, tissueor membrane fractions expressing the GPR17 receptor and/or functionalactive fragments thereof. In one embodiment of the invention, suchscreening assays may be provided, sold or offered in the form of a kitcomprising all or substantially all of the components to be used in theassay described herein.

One aspect of the present invention relates to a method of identifying atest compound's ability to modulate the GPR17 activity by contacting acompound with GPR17 (or a functional fragment thereof) in the presenceof a GPR17 agonist according to the present invention, and comparing theactivity of GPR17 in the presence of the agonist with and without thepresence of said test compound.

One aspect of the present invention relates to a method of treating oralleviating a GPR17 mediated disease, said method comprising (a) in afirst step identifying a GPR17 modulator, particularly an antagonist viathe screening methods and assays disclosed herein, and (b) in asubsequent step administering the GPR17 modulators identified in the1^(st) step (a) to a patient suffering from such a GPR 17 mediateddisease (such as spinal cord injury, multiple sclerosis, cerebral,cardiac and renal ischemia, and preferably stroke or multiplesclerosis), preferably in the form of a pharmaceutical compositioncomprising one or more pharmaceutically acceptable excipients.

One aspect of the present invention relates to GPR17 modulators,preferably antagonists, identified using the methods and screeningassays disclosed herein and methods for use of such modulators intherapy, particularly for the treatment or alleviation of spinal cordinjury, multiple sclerosis, cerebral, cardiac and renal ischemia, andpreferably of multiple sclerosis, ischemic diseases and stroke.

DESCRIPTION OF THE FIGURES

FIG. 1. Dose response curves (DRC) of compounds of formula I beingactive in the 1321N1 (A, B) and CHO cell systems (C, D) expressing GPR17recombinantly (n=3-29) determined from the calcium mobilization assaysof Example 2. For reference purposes the DRC of the lead compoundRA-II-150 is shown in all plots (closed red squares).

FIG. 2. Effect of RA-II-150, porphobilinogen (PBG) and 5-aminolevulinicacid (5-ALA) on forskolin-stimulated cAMP production, using the assay ofExample 3. Individual concentrations of RA-II-150 and PBG were tested inCHO-GPR17 (2A) and CHO cells (2B), respectively. Concentration-responsecurve of RA-II-150 in CHO-GPR17 (2C). Concentration-response curves of5-ALA ((A)—stock solved in DMSO, (B)—stock solved in water) in CHO-GPR17(2D), corrected for the effect seen in CHO) and CHO cells (2E). cAMPlevels induced by stimulation with forskolin (Fsk) were set 100%. Alldata are means (±s.e.m.) of two to six experiments performed induplicates. In both figures, the left data column corresponds to theuppermost legend and so forth.

FIG. 3. Concentration-effect curve of RA-II-150 in the ³⁵SGTPγS bindingassays of Example 4 on membranes from 1321N1-GPR17 astrocytoma cells.Data are means (±s.e.m.) of a representative experiment.

FIG. 4. Concentration-effect curve of RA-II-150 in the inositolphosphateIP1 assays of example 6 in HEK293 cells stably expressing hGPR17 taggedN-terminally with the triple HA epitope tag. Data are means (±s.e.m.) ofthree independent experiments.

FIG. 5. Concentration-effect curve of RA-II-150 in the β-arrestintranslocation assays of Example 5 using HEK293 cells co-expressing GPR17fused to Renilla Luciferase as energy donor and β-arrestin2 fused toGFP2 as energy acceptor. Data are means (±s.e.m.) of three independentexperiments

FIG. 6. Concentration-inhibition curve for a GPR17 antagonist,inhibiting functional activity of GPR17 stimulated with the EC₈₀ of thesmall molecule agonist RA-II-150 in the Ca²⁺+ mobilization assays ofExample 2 on CHO-GPR17 cells. Data are means (±s.e.m.) of fiveindependent experiments

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the compounds of formula I

-   -   and salts thereof, and methods for their use as further defined        herein,    -   wherein in formula I    -   R1 and R2 are independently selected from the group comprising        hydrogen, halogen, hydroxy, formyl, oxime, cyano, nitro, NR6R7,        carboxy, carbamoyl, (C₁-C₈)alkyl, (C₁-C₈) alkyloxy,        (C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyloxy, (C₃-C₈)cycloalkylamino,        aryl, heteroaryl, aryloxy, heteroaryloxy, halogen,        trifluoromethyl, (C₁-C₈)alkylcarbonyl, (C₁-C₈)        alkylaminocarbonyl, di(C₁-C₈)alkylaminocarbonyl, arylcarbonyl,        heteroarylcarbonyl, aryl(C₁-C₈)alkyl, heteroaryl(C₁-C₈)alkyl,        aryl(C₁-C₈)alkyloxy, heteroaryl(C₁-C₈)alkyloxy,        aryl(C₁-C₈)alkylcarbonyl, heteroaryl(C₁-C₈)alkylcarbonyl,        aryl(C₁-C₈)alkyloxycarbonyl, heteroaryl(C₁-C₈)alkyloxycarbonyl,        (C₁-C₈)alkyloxycarbonyl, (C₁-C₈)alkylsulfonyl, arylsulfonyl,        heteroarylsulfonyl, sulfamoyl, sulfonylamino,        (C₁-C₈)alkylaminosulfonyl, di(C₁-C₈)alkylaminosulfonyl,        arylsulfonylamino, heteroarylsulfonylamino and        (C₁-C₈)alkylsulfonylamino; wherein each alkyl, alkenyl, alkynyl        or cycloalkyl may be unsubstituted or substituted with one or        more residues selected from among hydroxyl, (C₁-C₅)alkyloxy,        (C₁-C₃)alkyloxy(C₁-C₃)alkyloxy, halogen, and NR6R7; and wherein        each aryl or heteroaryl can be unsubstituted or substituted with        one or more residues selected from among hydroxyl,        (C₁-C₅)alkyloxy, halogen, (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl,        carboxy, NR6R7, cyano, trifluormethyl and nitro;    -   R3 is selected from hydrogen, a group —(CH₂)_(m)CH₂—COOH, OH,        NH, and (C₁-C₅)alkyl which is optionally substituted with one or        more halogens, one or two hydroxyl groups or (C₁-C₃)alkoxy;    -   R4 is hydrogen or fluoro, and is preferably hydrogen;    -   R5 is selected from hydrogen, halogen, (C₁-C₃)alkyl,        (C₁-C₃)alkyloxy, (C₁-C₃)alkylthio, (C₂-C₄)alkenyl,        (C₂-C₄)alkynyl, and NR6R7;    -   R6 and R7 are independently selected from hydrogen,        (C₁-C₆)alkyl, (C₃-C₇)cycloalkyl, phenyl, heteroaryl,        phenyl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfonyl, phenylsulfonyl, heteroarylsulfonyl,        (C₁-C₈)alkylcarbonyl, (C₁-C₈)alkoxycarbonyl, aminocarbonyl,        (C₁-C₆)alkylaminocarbonyl, phenylcarbonyl, and        heteroarylcarbonyl; wherein each alkyl may be unsubstituted or        substituted with one or more residues selected from among        hydroxyl, (C₁-C₃)alkyloxy, phenyl, halo, carboxy, and NR8R9; and        wherein R6 and R7 may form a 5- to 7-membered cycle; and wherein        phenyl or heteroaryl can be unsubstituted or substituted with        one or more residues selected from among hydroxyl,        (C₁-C₃)alkyloxy, halogen, (C₁-C₃)alkyl, carboxy, NR8R9, cyano,        trifluormethyl and nitro;    -   R8 and R9 are independently selected from among hydrogen and        (C₁-C₃)alkyl;    -   n and m are independently 0, 1 or 2.

In one preferred aspect, R1 in formula I is not hydrogen.

In one aspect, R1 and R2 are independently selected from the groupcomprising hydrogen, halogen, hydroxyl, cyano, nitro, NR6R7, carboxy,(C₁-C₅)alkyl, (C₁-C₅) alkyloxy, (C₁-C₅)alkylthio, (C₂-C₅)alkenyl,(C₂-C₅)alkynyl, (C₃-C₆)cycloalkyl, (C₃-C₆)cycloalkyloxy,(C₃-C₆)cycloalkylamino, phenyl, C₅-C₆heteroaryl, phenyloxy C₅-C₆heteroaryloxy, halogen, trifluoromethyl, (C₁-C₅)alkylcarbonyl, (C₁-C₅)alkylaminocarbonyl, di(C₁-C₅)alkylaminocarbonyl, phenylcarbonyl,heteroarylcarbonyl, phenyl(C₁-C₃)alkyl, heteroaryl(C₁-C₃)alkyl,phenyl(C₁-C₃)alkyloxy, heteroaryl(C₁-C₃)alkyloxy,phenyl(C₁-C₃)alkylcarbonyl, heteroaryl(C₁-C₃)alkylcarbonyl,phenyl(C₁-C₃)alkyloxycarbonyl, heteroaryl(C₁-C₃)alkyloxycarbonyl,(C₁-C₅)alkyloxycarbonyl, (C₁-C₅)alkylsulfonyl, phenylsulfonyl,heteroarylsulfonyl, sulfamoyl, sulfonylamino, (C₁-C₅)alkylaminosulfonyl,di(C₁-C₅)alkylaminosulfonyl, phenylsulfonylamino,heteroarylsulfonylamino and (C₁-C₅)alkylsulfonylamino; wherein eachalkyl, alkenyl, alkynyl or cycloalkyl may be unsubstituted orsubstituted with one or more residues selected from among hydroxyl,(C₁-C₃)alkyloxy, (C₁-C₃)alkyloxy(C₁-C₃)alkyloxy, halogen, and NR6R7; andwherein each phenyl or heteroaryl can be unsubstituted or substitutedwith one or more residues selected from among hydroxyl, (C₁-C₃)alkyloxy,halogen, (C₁-C₃)alkyl, (C₃-C₆)cycloalkyl, carboxy, NR6R7, cyano,trifluormethyl and nitro, wherein R1 is not hydrogen.

In one preferred aspect, R5 is hydrogen, halogen, methyl or methoxy. Inone aspect, R5 is fluoro or hydrogen, and particularly hydrogen.

In one aspect, R6 and R7 are independently selected from hydrogen,(C₁-C₄)alkyl, (C₃-C₇)cycloalkyl, phenyl, heteroaryl, phenyl(C₁-C₃)alkyl,heteroaryl(C₁-C₃)alkyl, (C₁-C₃)alkylsulfonyl, phenylsulfonyl,heteroarylsulfonyl, (C₁-C₃)alkylcarbonyl, (C₁-C₃)alkoxycarbonyl,aminocarbonyl, (C₁-C₃)alkylaminocarbonyl, phenylcarbonyl, andh(C₅-C₆)eteroarylcarbonyl; wherein each alkyl may be unsubstituted orsubstituted with one or more residues selected from among hydroxyl,methoxy, phenyl, fluoro, chloro, bromo, carboxy, and NR8R9; and whereinR6 and R7 may form a 5- to 7-membered cycle; and wherein phenyl orheteroaryl can be unsubstituted or substituted with one or more residuesselected from among hydroxyl, (C₁-C₃)alkyloxy, halogen, (C₁-C₃)alkyl,carboxy, NR8R9, cyano, trifluormethyl and nitro.

One aspect of the invention relates to compounds of formula I and saltsthereof, and methods for their use as further defined herein, wherein

R1 is selected from fluoro, chloro, bromo, iodo, (C₁-C₃)alkyl,(C₁-C₃)alkoxy, phenyl, phenyl (C₁-C₃)alkyl, phenyl(C₁-C₃)alkoxy,phenylcarbonyl, (C₅-C₆)heteroaryl, (C₅-C₆)heteroarylcarbonyl,(C₅-C₆)heteroaryl(C₁-C₃)alkyl and (C₅-C₆)heteroaryl(C₁-C₃)alkoxy,wherein the alkyl and alkoxy groups are optionally substituted with oneor more halogens, (C₁-C₃)alkoxy, or hydroxyl, and wherein the phenyl and(C₅-C₆)heteroaryl groups can be substituted with one or more halogens,(C₁-C₃)alkoxy, (C₁-C₃)alkyl, NR6R7, or hydroxyl;R2 is selected from hydrogen, fluoro, chloro, bromo, iodo, (C₁-C₃)alkyl,(C₁-C₃)alkoxy, and phenyl, wherein the alkyl and alkoxy groups areoptionally substituted with one or more halogens, (C₁-C₃)alkoxy, orhydroxyl, and wherein the phenyl group can be substituted with one ormore halogens, (C₁-C₃)alkoxy, (C₁-C₃)alkyl, or hydroxyl,R3 is selected from hydrogen and a group —(CH₂)_(m)CH₂—COOH;R4 is hydrogen or fluoro, and is preferably hydrogen;R5 is selected from hydrogen, halogen, methyl or methoxy, and preferablyrepresents fluoro or hydrogen, and particularly hydrogen;n and m are independently 0, 1 or 2.

One preferred aspect of the invention relates to compounds of formula Iand salts thereof, and methods of their use as further defined herein,wherein

R1 is selected from halogen, (C₁-C₃)alkyl, (C₁-C₃)alkoxy, phenyl and(C₅-C₆)heteroaryl, wherein the alkyl and alkoxy groups are optionallysubstituted with one or more halogens, and wherein the phenyl and(C₅-C₆)heteroaryl groups are optionally substituted with halogen,(C₁-C₃)alkyl or (C₁-C₃)alkoxy;R2 is selected from hydrogen, halogen, (C₁-C₃)alkyl, (C₁-C₃)alkoxy, andphenyl, wherein the alkyl and alkoxy groups are optionally substitutedwith one or more halogens, and wherein the phenyl group is optionallysubstituted with halogen, methyl or methoxy;R3 is selected from hydrogen and a group —(CH₂)_(m)CH₂—COOH;R4 is selected from hydrogen and fluoro, preferably hydrogen,R5 is selected from hydrogen and fluoro, preferably hydrogen;n and m are independently 0, 1 or 2.

In one preferred aspect, in the compounds of formula I and their saltsfor use as defined herein,

R1 is selected from the group consisting of methyl, CF₃, chloro, fluoro,bromo, iodo, phenyl and (C₅-C₆)heteroaryl, wherein the phenyl orheteroaryl group is optionally substituted with halogen, methyl ormethoxy;

R2 is selected from the group consisting of hydrogen, methyl, CF₃,chloro, fluoro, bromo, iodo and phenyl;

R3 is hydrogen, carboxymethyl, or carboxyethyl;

R4 and R5 are both hydrogen; and

n is 1.

Preferred compounds of formula I and their salts are those wherein,

R1 is selected from the group consisting of methyl, methoxy, hydroxy,CF₃, chloro, fluoro, bromo, iodo, thienyl, furanyl, pyridyl, and phenylwhich is optionally substituted with halogen, methyl or methoxy;

R2 is selected from the group consisting of hydrogen, methyl, methoxy,hydroxyl, CF₃, chloro, fluoro, bromo, iodo and phenyl;

R3 is hydrogen, carboxymethyl, or carboxyethyl;

R4 and R5 are independently fluoro or, preferably, hydrogen; and

n is 1.

Particularly preferred compounds for use in the methods of the presentinvention are 3-(2-carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylicacid,3-(2-carboxyethyl)-4,6-dichloro-(1-carboxyethyl)-indole-2-carboxylicacid, 3-(2-carboxyethyl)-4,6-dimethyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-chloro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-difluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dibromo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-bromo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-iodo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-diiodo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-diphenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-phenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6,7-dichloro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-chloro-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-bromo-2-carboxy-7-fluoro-1H-indole-2-carboxylicacid, 3-(2-carboxyethyl)-4,6-dimethoxy-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-phenoxy-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-benzyl-2-carboxy-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dihydroxy-1H-indole-2-carboxylic acid,3-(2-Carboxyethyl)-6-(4-fluorophenyl)-1H-indol-2-carboxylic acid,3-(2-carboxyethyl)-6-furanyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-thienyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-7-fluoro-6-phenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-(4-fluorophenyl)-7-fluoro-1H-indol-2-carboxylicacid, 3-(2-carboxyethyl)-6-furanyl-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-thienyl-7-fluoro-1H-indole-2-carboxylic acid, andsalts thereof.

One aspect relates to a novel compound selected from3-(2-carboxyethyl)-4,6-dibromo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dichloro-(1-carboxyethyl)-indole-2-carboxylicacid, 3-(2-carboxyethyl)-6-bromo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-iodo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-diiodo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-diphenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-phenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6,7-dichloro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-chloro-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-bromo-2-carboxy-7-fluoro-1H-indole-2-carboxylicacid, 3-(2-carboxyethyl)-6-benzyl-2-carboxy-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dihydroxy-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-(4-fluorophenyl)-1H-indol-2-carboxylic acid,3-(2-carboxyethyl)-6-furanyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-thienyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-7-fluoro-6-phenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-(4-fluorophenyl)-7-fluoro-1H-indol-2-carboxylicacid, 3-(2-carboxyethyl)-6-furanyl-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-thienyl-7-fluoro-1H-indole-2-carboxylic acid, andsalts thereof. Another aspect relates to methods of using said novelcompounds in therapy.

In one aspect, the GPR agonists of the present invention can also beused in methods and assays to be used for the identification of otherGPR 17 modulators, particularly of GPR 17 antagonists.

One aspect of the present invention relates to a method of identifying acompound that modulates GPR 17 activity, said method (hereafter with orwithout its various aspects disclosed herein also called “the screeningmethod of the present invention”) comprising the steps of

-   -   (a) contacting a test compound with GPR17, or a functional GPR17        fragment in the presence of a suitable amount of a GPR17 agonist        of formula I

-   -   -   or a salt thereof

    -   (b) determining the biological activity of GPR17 or said        functional GPR17 fragment after the addition of said test        compound, and

    -   (c) comparing the biological activity determined in step (b)        with the activity of GPR17 or said functional GPR17 fragment in        the presence of said GPR 17 agonist of formula I without the        addition of said test compound,

    -   wherein in formula I

    -   R1 and R2 are independently selected from the group comprising        hydrogen, halogen, hydroxy, formyl, oxime, cyano, nitro, amino,        NR6R7, carboxy, carbamoyl, (C₁-C₈)alkyl, (C₁-C₈) alkyloxy,        (C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyloxy, (C₃-C₈)cycloalkylamino,        aryl, heteroaryl, aryloxy, heteroaryloxy, halogen,        trifluoromethyl, (C₁-C₈)alkylcarbonyl, (C₁-C₈)        alkylaminocarbonyl, di(C₁-C₈)alkylaminocarbonyl, arylcarbonyl,        heteroarylcarbonyl, aryl(C₁-C₈)alkyl, heteroaryl(C₁-C₈)alkyl,        aryl(C₁-C₈)alkyloxy, heteroaryl(C₁-C₈)alkyloxy,        aryl(C₁-C₈)alkylcarbonyl, heteroaryl(C₁-C₈)alkylcarbonyl,        aryl(C₁-C₈)alkyloxycarbonyl, heteroaryl(C₁-C₈)alkyloxycarbonyl,        (C₁-C₈)alkyloxycarbonyl, (C₁-C₈)alkylsulfonyl, arylsulfonyl,        heteroarylsulfonyl, sulfamoyl, sulfonylamino,        (C₁-C₈)alkylaminosulfonyl, di(C₁-C₈)alkylaminosulfonyl,        arylsulfonylamino, heterosulfonylamino and alkylsulfonylamino;        wherein each alkyl, alkenyl, alkynyl or cycloalkyl may be        unsubstituted or substituted with one or more residues selected        from among hydroxyl, (C₁-C₈)alkyloxy,        (C₁-C₃)alkyloxy(C₁-C₃)alkyloxy, halogen, and NR6R7; and wherein        each aryl or heteroaryl can be unsubstituted or substituted with        one or more residues selected from among hydroxyl,        (C₁-C₅)alkyloxy, halogen, (C₁-C₅)alkyl, (C₃-C₈)cycloalkyl,        carboxy, NR6R7, cyano, trifluormethyl and nitro;

    -   R3 is selected from hydrogen, a group —(CH₂)_(m)CH₂—COOH, OH,        NH, and (C₁-C₅)alkyl which is optionally substituted with one or        more halogens, one or two hydroxyl groups or methoxy,

    -   R4 is selected from hydrogen and fluoro;

    -   R5 is selected from hydrogen, halogen, (C₁-C₃)alkyl,        (C₁-C₃)alkyloxy, (C₁-C₃)alkylthio, (C₂-C₄)alkenyl,        (C₂-C₄)alkynyl, and NR6R7;

    -   R6 and R7 are independently selected from hydrogen,        (C₁-C₆)alkyl, (C₃-C₇)cycloalkyl, phenyl, heteroaryl,        phenyl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfonyl, phenylsulfonyl, heteroarylsulfonyl,        (C₁-C₆)alkylcarbonyl, (C₁-C₆)alkoxycarbonyl, aminocarbonyl,        (C₁-C₆)alkylaminocarbonyl, phenylcarbonyl, and        heteroarylcarbonyl; wherein each alkyl may be unsubstituted or        substituted with one or more residues selected from among        hydroxyl, (C₁-C₃)alkyloxy, phenyl, halo, carboxy, and NR8R9; and        wherein R6 and R7 may form a 5- to 7-membered cycle; and wherein        phenyl or heteroaryl can be unsubstituted or substituted with        one or more residues selected from among hydroxyl,        (C₁-C₃)alkyloxy, halogen, (C₁-C₃)alkyl, carboxy, NR8R9, cyano,        trifluormethyl and nitro;

    -   R8 and R9 are independently selected from hydrogen and        (C₁-C₃)alkyl;

    -   n and m are independently 0, 1 or 2.

    -   Preferred compounds for use in the screening method of the        present invention are disclosed further above and in the claims.

    -   Another aspect of the present invention relates to a screening        assay for the identification of GPR 17 modulators, preferably of        GPR antagonists among a multitude of compounds, said screening        assay (hereafter with or without its various aspects disclosed        herein also called “assay of the present invention”) comprising        -   (a) cells or membrane fractions expressing GPR17 or a            functional GPR17 fragment and            -   (b) a suitable amount of a GPR17 agonist of formula I

-   -   or a salt thereof, wherein in formula I    -   R1 and R2 are independently selected from the group comprising        hydrogen, halogen, hydroxy, formyl, oxime, cyano, nitro, amino,        NR6R7, carboxy, carbamoyl, (C₁-C₈)alkyl, (C₁-C₈) alkyloxy,        (C₁-C₈)alkylthio, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkyloxy, (C₃-C₈)cycloalkylamino,        aryl, heteroaryl, aryloxy, heteroaryloxy, halogen,        trifluoromethyl, (C₁-C₈)alkylcarbonyl, (C₁-C₈)        alkylaminocarbonyl, di(C₁-C₈)alkylaminocarbonyl, arylcarbonyl,        heteroarylcarbonyl, aryl(C₁-C₈)alkyl, heteroaryl(C₁-C₈)alkyl,        aryl(C₁-C₈)alkyloxy, heteroaryl(C₁-C₈)alkyloxy,        aryl(C₁-C₈)alkylcarbonyl, heteroaryl(C₁-C₈)alkylcarbonyl,        aryl(C₁-C₈)alkyloxycarbonyl, heteroaryl(C₁-C₈)alkyloxycarbonyl,        (C₁-C₈)alkyloxycarbonyl, (C₁-C₈)alkylsulfonyl, arylsulfonyl,        heteroarylsulfonyl, sulfamoyl, sulfonylamino,        (C₁-C₈)alkylaminosulfonyl, di(C₁-C₈)alkylaminosulfonyl,        arylsulfonylamino, heterosulfonylamino and alkylsulfonylamino;        wherein each alkyl, alkenyl, alkynyl or cycloalkyl may be        unsubstituted or substituted with one or more residues selected        from among hydroxyl, (C₁-C₅)alkyloxy,        (C₁-C₃)alkyloxy(C₁-C₃)alkyloxy, halogen, and NR6R7; and wherein        each aryl or heteroaryl can be unsubstituted or substituted with        one or more residues selected from among hydroxyl,        (C₁-C₅)alkyloxy, halogen, (C₁-C₅)alkyl, (C₃-C₈)cycloalkyl,        carboxy, NR6R7, cyano, trifluormethyl and nitro;    -   R3 is selected from hydrogen, a group —(CH₂)_(m)CH₂—COOH, OH,        NH, and (C₁-C₅)alkyl which is optionally substituted with one or        more halogens, one or two hydroxyl groups or (C₁-C₃)alkoxy,    -   R4 is selected from hydrogen and fluoro, and is preferably        hydrogen;    -   R5 is selected from hydrogen, halogen, (C₁-C₃)alkyl, (C₁-C₃)        alkyloxy, (C₁-C₃)alkylthio, (C₂-C₄)alkenyl, (C₂-C₄)alkynyl, and        NR6R7,    -   R6 and R7 are independently selected from hydrogen,        (C₁-C₆)alkyl, (C₃-C₇)cycloalkyl, phenyl, heteroaryl,        phenyl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl,        (C₁-C₆)alkylsulfonyl, phenylsulfonyl, heteroarylsulfonyl,        (C₁-C₆)alkylcarbonyl, (C₁-C₆)alkoxycarbonyl, aminocarbonyl,        (C₁-C₆)alkylaminocarbonyl, phenylcarbonyl, and        heteroarylcarbonyl; wherein each alkyl may be unsubstituted or        substituted with one or more residues selected from among        hydroxyl, (C₁-C₃)alkyloxy, phenyl, halo, carboxy, and NR8R9; and        wherein R6 and R7 may form a 5- to 7-membered cycle; and wherein        phenyl or heteroaryl can be unsubstituted or substituted with        one or more residues selected from among hydroxyl,        (C₁-C₃)alkyloxy, halogen, (C₁-C₃)alkyl, carboxy, NR8R9, cyano,        trifluormethyl and nitro;    -   R8 and R9 are independently selected from among hydrogen and        (C₁-C₃)alkyl;    -   n and m are independently 0, 1 or 2.    -   Preferred compounds for use in the assay of the present        invention are disclosed further above and in the claims.

DEFINITIONS

The term “GPR 17” as used herein means a polypeptide showing GPR 17activity, namely a G protein-coupled receptor having Uracilnucleotide/cysteinyl leukotriene receptor activity.

Preferably, the term “GPR17” as used herein includes but is not limitedto the human short splicing variant of GPR 17 (SEQ ID NO1), the humanlong splicing variant of GPR 17 (SEQ ID NO 2), the rat GPR 17 (SEQ ID NO3), the mouse GPR 17 (SEQ ID NO 4), and any other natural occurring GPR17.

Further, the term also refers to functional fragments, fractions orsubsequences of the above discussed polypeptides.

Even more preferred, said polypeptide is at least one selected from thegroup consisting of

-   -   (a) a polypeptide having the amino acid sequence of SEQ ID NO 1        or SEQ ID NO 2,    -   (b) polypeptides comprising the amino acid sequence of SEQ ID NO        1 or SEQ ID NO 2,    -   (c) polypeptides having at least about 60%, preferably at least        about 70%, more preferably at least about 75%, even more        preferably at least 80%, at least about 85%, at least about 90%,        at least about 95%, or even at least about 98% sequence identity        to SEQ ID NO 1 or SEQ ID NO 2, wherein the sequence identity is        preferably determined with one of the methods discussed below,    -   (d) functional fragments, fractions or subsequences of any of        the sequences discussed in (a), (b) or (c), and/or    -   (e) fusion products which have been obtained after deletion of a        fragment, fraction or subsequence from any of the sequences        discussed in (a), (b), (c) or (d), and fusion of the remaining        sequences.

The sequences set forth under (a)-(e) will also be called “querysequences” in the following.

A “functional fragment, fraction or subsequence” comprises a polypeptidewhich represents a part of the amino acid sequence of GPR17 as furtherdefined herein, wherein said functional GPR 17 fragment still showsGPR17 activity. One example of a functional GPR 17 fragment can be atruncated GPR 17, wherein a certain number of amino acids is missing atthe N′ and/or C′ terminus of GPR17. Another example of a functional GPR17 fragment is one of various subsequent parts of a “splitted” GPR 17receptor, wherein said GPR17 parts are contained on individualexpression plasmids and are physiologically inactive when expressedalone, but assemble to a functional GPR 17 receptor in an appropriateenvironment upon co-transfection of cells. Methods for the expression of“split receptors” are disclosed e.g. in Maggio R et al, FEBS Lett. 1993Mar. 15; 319(1-2):195-200, and Schöneberg T et al, J Biol Chem. 1995Jul. 28; 270(30):18000-6.

The term “% sequence identity” as used herein refers to the identity ofone sequence (e.g., sequence A) to another sequence (e.g., sequence B)over the whole length of either of the sequences, i.e., to thepercentage of residues that are identical in the two sequences to becompared. In general, the two sequences are aligned to give a maximumcorrelation between the sequences. If applicable, this may includeinserting “gaps” in either one or both sequences, to enhance the degreeof alignment.

Further, sequence alignment generally falls into two categories: globalalignments and local alignments. Calculating a global alignment is aform of global optimization that “forces” the alignment to span theentire length of all query sequences. By contrast, local alignmentsidentify regions of similarity within long sequences that are oftenwidely divergent overall. In the present invention, either type ofalignment can be performed when “% sequence identity” is determined.

Readily available computer programs can be used to aid in the analysis,such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and StructureM. O. Dayhoff ed.,* 5 Suppl. 3:353-358, National biomedical ResearchFoundation, Washington, D.C.

The preferred way to determine “% sequence identity” between twosequences is to count the exact number of matches between two sequencesaligned as discussed above, dividing by the length of the shortersequence, and multiplying the result by 100. This approach is a merelyarithmetical approach which does not provide any kind of biologicalweighing.

Another approach to determine the “% sequence identity” of two sequenceswhich is likewise preferred is the GenePAST™ algorithm provided byGenomeQuest, Inc. This algorithm was formerly called “Kerr”, and isdiscussed, e.g., in Dufresne et al, Nature Biotechnology 20, 1269-1271(2002), or Andree et al, World Patent Information, 2008, vol. 30, issue4, pages 300-308. Again, this approach carries out a merely arithmeticaldetermination of “% sequence identity” without any biological weighing.

In an exemplary case where (i) the query sequence has 339 residues (likeSEQ ID No 1, which defines one possible GPR 17 polypeptide according tothe invention), (ii) the subject sequence has 400 residues, and (iii) analignment of 200 residues was found between both sequences with 4mismatches comprised in the alignment (which means that a functionalfragment of query sequences aligns with the subject sequence), thefollowing approaches can be used to determine the “% sequence identity”in accordance with the present invention:

-   -   1. In a preferred embodiment, the “% sequence identity” is        calculated over the length of the entire query sequence (e.g.        339 residues). In this case, 143 mismatches have to be        considered, which leads to a % sequence identity of        (339−143)/339=57.8%    -   2. In another preferred embodiment which is used when a fragment        of the query sequence is to be compared with a given subject        sequence, the “% sequence identity” is calculated over the        alignment length of e.g. 200 residues (e.g., the functional        fragment of the query sequence which aligns with the subject        sequence serves as a basis for sequence identity calculation).        In this case, both sequences share a % sequence identity of        (200−4)/200=98%    -   3. In yet another embodiment, the “% sequence identity” can be        calculated over the length of the entire subject sequence (400        residues), 204 mismatches have to be considered, which leads to        a % sequence identity of (400−204)/400=49%. This option, which        takes the subject sequence as a basis for sequence identity        calculation, is however less preferred.

An alternative method often used to determine the “% sequence identity”between two polypeptide sequences, and which shall also be considered asan alternative means to determine % sequence identity according to thepresent invention, uses the BLAST algorithm (Tatusova and Madden, FEMSMicrobiol Lett 174, 1999, 247; Altschul, et al, J. Mol. Biol.215:403-410 (1990)), which is available e.g. through the National Centerfor Biotechnology Information (NCBI; “blastp”), National Library ofMedicine, National Institutes of Health, Building 38A, 8600 RockvillePike, Bethesda, Md. 30 20894, USA (Wheeler et al., Nucleic Acid Res 35,2007, D5; Pearson, Methods Enzymol 183, 1990, 63).

However, The BLAST algorithm performs alignment scoring adjustmentsbased on considerations of biological relevance between query andsubject sequences. This involves the risk of mismatches between merearithmetic sequence comparison, as preferred under the above mentioned“% sequence identity” language, and biologically weighted sequencecomparison, as performed, e.g., by BLAST. However, as the BLASTalgorithm is frequently used, finds wide acceptance and is publiclyavailable, it can as well be used in the context of the presentinvention to determine “% sequence identity”.

Other algorithms which can also be used to determine “% sequenceidentity” within the meaning of the present invention comprise, but arenot restricted to Smith & Waterman (Smith and Waterman, Journal ofMolecular Biology 147: 195-197 and BLW (also known as “FragmentSearch”).

In a preferred embodiment, the “polypeptide showing GPR 17 activity”shows at least about 20%, more preferably at least about 30%, morepreferably at least about 40%, at least about 50%, at least about 60%,at least about 70%, and even more preferably at least about 80% or atleast about 90% of the activity of the human GPR 17 receptor having theamino acid sequence of SEQ No1.

The term “identifying a compound that modulates GPR 17 activity”includes determining the GPR17 activity (either wanted or unwanted) of aparticular compound, as well as the screening of a multitude ofcompounds for the identification of GPR 17 antagonists showing a certainthreshold of GPR 17 activity, and may also include the confirmatorytesting of already known or suggested GPR17 functional activities of agiven compound. The term “identifying a compound that modulates GPR 17activity” also includes the determination or comparison of a testcompound's functional activity towards GPR17 in a particularexperimental setting, such as e.g. in a particular cell line, in a newexperimental setup, or in cells or tissues from a particular diseasestate.

The term “screening assay” or “screening method” as used herein refersto an assay or method which can be used and/or adapted to determineand/or analyze the GPR17 modulating properties of a single compoundand/or of a variety or large number of compounds, such as e.g. in a highthroughput screening format.

-   -   “Alkyl” includes monovalent saturated aliphatic hydrocarbyl        groups. The hydrocarbon chain may be either straight-chained or        branched. “Alkyl” has preferably 1-8 carbon atoms        (“(C₁-C₈)alkyl”) or 1-6 carbon atoms (“(C₁-C₆)alkyl”), and in        some instances even more preferably 1-5 carbon atoms        (“(C₁-C₅)alkyl”), 1-4 carbon atoms (“(C₁-C₄)alkyl”), or only 1-3        carbon atoms (“(C₁-C₃)alkyl”). This term is exemplified by        groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,        iso-butyl, tert-butyl, t-amyl, and the like.    -   “Alkylsulfonyl” includes a radical-S(O)₂R, wherein R is an alkyl        group as defined herein. Representative examples include, but        are not limited to, methanesulfonyl, ethylsulfonyl,        propylsulfonyl, butylsulfonyl and the like.    -   “Alkylthio” includes a radical-S—R wherein R is an alkyl group        as defined herein that may be optionally substituted as defined        herein. Representative examples include, but are not limited to,        methylthio, ethylthio, propylthio, butylthio, and the like.    -   “Alkylaminosulfonyl” includes the group —SO₂—NH-Alkyl, wherein        “alkyl” is preferably selected from the groups specified in the        definition of “alkyl” further above. Examples of        “alkylaminosulfonyl” are e.g. methylaminosulfonyl,        ethylaminosulfonyl or butylaminosulfonyl.    -   “Dialkylaminosulfonyl” includes the group —SO₂—N-dialkyl,        wherein each “alkyl” is preferably and independently selected        from the groups specified in the definition of “alkyl” further        above. Examples of “alkylaminosulfonyl” are e.g.        N,N-dimethylaminosulfonyl, N,N-methylethylaminosulfonyl or        N,N-methylbutylaminosulfonyl.    -   “Alkylsulfonylamino” includes the group —NH—SO₂-Alkyl, wherein        alkyl is preferably selected from the groups specified in the        definition of “alkyl” further above. Most preferably “alkyl” in        “alkylsulfonylamino” is aC₁-C₈-alkylgroup, such as e.g.        methanesulfonylamino.    -   “Alkylcarbonyl” includes the group —C(O)-alkyl, wherein alkyl is        preferably selected from the groups specified in the definition        of “alkyl” further above. “Alkylcarbonyl” is particularly        preferably —C(O)—C₁-C₆-Alkyl, and most preferably acetyl,        propionyl oder butyryl.    -   “Alkylaminocarbonyl” includes the groups —C(O)—NH-alkyl wherein        “alkyl” is preferably selected from the groups specified in the        definition of “alkyl” further above. “Alkylaminocarbonyl” is        particularly preferably —C(O)—NH—(C₁-C₆)Alkyl    -   “Dialkylaminocarbonyl” includes the group —CO—N-dialkyl, wherein        each “alkyl” is preferably and independently selected from the        groups specified in the definition of “alkyl” further above.        “Dialkylaminocarbonyl” is particularly preferably        —C(O)—N-di(C₁-C₆)alkyl    -   “Alkyloxy” or “alkoxy” includes the group —OR wherein R is        “alkyl” as defined further above. Particular alkyloxy groups        include, by way of example, methoxy, ethoxy, n-propoxy,        isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy,        1,2-dimethylbutoxy, and the like.    -   “Alkyloxyalkyloxy” refers to the group —OROR′, wherein R and R′        are the same or different “alkyl” groups as defined further        above.    -   “Alkyloxycarbonyl” refer to the radical —C(═O)—O—R, wherein R is        an alkyl group as defined herein.    -   “Alkenyl” includes monovalent olefinically unsaturated        hydrocarbyl groups being straight-chained or branched and having        at least 1 double bond. “Alkenyl” has preferably 2-8 carbon        atoms (“C₂-C₈ alkenyl”) more preferably 2-6 carbon atoms        (“(C₂-C₆)alkenyl”), and in some instances even more preferably        2-5 carbon atoms (“(C₂-C₅)alkenyl”), 2-4 carbon atoms        (“(C₂-C₄)alkenyl”), or only 2-3 carbon atoms (“(C₂-C₃)alkenyl”).        Particular alkenyl groups include ethenyl (—CH═CH2), n-propenyl        (—CH2CH═CH2), isopropenyl (C(CH3)=CH2), and the like.    -   “Alkynyl” includes unsaturated hydrocarbyl groups being        straight-chained or branched and having at least 1 triple bond.        “Alkynyl” has preferably 2-8 carbon atoms (“(C₂-C₈)alkynyl”)        more preferably 2-6 carbon atoms (“(C₂-C₆)alkynyl”), and in some        instances even more preferably 2-5 carbon atoms        (“(C₁-C₅)alkynyl”), 2-4 carbon atoms (“(C₂-C₄)alkynyl”), or only        2-3 carbon atoms (“(C₂-C₃)alkynyl”). A preferred alkynyl group        is ethynyl (acetylenyl).    -   “Amino” refers to the radical-NH₂.    -   “Aryl” refers to an aromatic hydrocarbyl radical. Examples of        “aryl” radicals are phenyl, naphthyl, indenyl, azulenyl,        fluorine or anthracene, wherein phenyl is preferred. “Arylalkyl”        comprises the group -alkyl-aryl, wherein “aryl” and “alkyl” have        the meaning as defined further above. Examples of arylalkyl        groups are phenylpropyl, phenylethyl and benzyl, wherein benzyl        is a particularly preferred arylalkyl group.    -   “Arylalkyloxy” comprises the group —O-alkyl-aryl, wherein “aryl”        and “alkyl” have the meaning as defined further above, and        wherein aryl is preferably phenyl. Examples of arylalkyloxy        groups are pehnylethyloxy and benzyloxy.    -   “Aryloxy” comprises the group-O-aryl, wherein aryl” has the        meaning as defined further above, and wherein aryl is preferably        phenyl    -   “Arylcarbonyl” is —C(O)-aryl, wherein aryl” has the meaning as        defined further above, and wherein aryl is preferably phenyl    -   “Arylalkylcarbonyl” is —C(O)-alkyl-aryl, wherein “aryl” and        “alkyl” have the meaning as defined further above, and wherein        aryl is preferably phenyl    -   “Arylalkyloxycarbonyl” is the group —C(O)—O-alkyl-aryl, wherein        “aryl” and “alkyl” have the meaning as defined further above,        and wherein aryl is preferably phenyl    -   “Arylsulfonyl” is —SO₂-aryl, wherein aryl” has the meaning as        defined further above, and wherein aryl is preferably phenyl    -   “Arylsulfonylamino” refers to the group —NH—SO2-aryl, wherein        “aryl” has the meaning as defined further above, and wherein        aryl is preferably phenyl    -   “Carbamoyl” refers to the group —C(═O)NH2    -   “Carboxy” refers to the radical —C(═O)OH.    -   “Cycloalkyl” refers to cyclic saturated aliphatic hydrocarbyl        groups. The numbers of C-atoms referenced in connection with a        given cycloalkyl group corresponds to the number of ring forming        carbon atoms, e.g. “(C₃-C₆)cycloalkyl” refers to a cycloalkyl        with between three and six ring-forming C atoms. Examples of        “cycloalkyl” are (C₃-C₈)cycloalkyls, (C₃-C₇)cycloalkyls, or more        specifically (C₃-C₆)cycloalkyls such as e.g. cyclopropyl,        cyclobutyl, cyclopentyl, cyclohexyl etc. If a “cycloalkyl”        carries more than one substituent, e.g. one or more alkyl        substituent these substituents may be attached to the same or to        different ring-forming carbon atoms.    -   “Cycloalkyloxy” refers to the group —OR, wherein R is        “cycloalkyl” group as defined further above.    -   “Cycloalkylamino” refers to the group —NHR, wherein R is        “cycloalkyl” group as defined further above.    -   “Cyano” refers to the radical —C≡N.    -   “Formyl” refers to the group —C(═O)H    -   “Halo” or “halogen” refers to fluoro, chloro, bromo and iodo.    -   “Heteroaryl” refers to aromatic ring system containing at least        one heteroatom such as O, S or N. Examples of heteroaryl        radicals are furanyl, thienyl, pyrollyl, thiazolyl, oxazolyl,        imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,        oxadiazolyl, thiadiazolyl, pyranyl, pyridinyl, pyridazinyl,        pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, indolinyl,        indolyl, isoindolyl, benzofuranyl, benzothiophenyl,        benzoimidazolyl, benzthiazolyl, purinyl, quinazolinyl,        quinolinyl, isoquinolinyl, quinolizinyl, pteridinyl, carbazolyl,        wherein one-ring systems, in particular one ring-systems with 5        to 6 ring atoms (“C₅-C₆ heteroaryl”) such as e.g. pyridinyl,        thienyl, oxazolyl, triazolyl, pyrimidinyl, imidazolyl, and the        like are preferred.    -   “Heteroarylalkyl” comprises the group -alkyl-heteroaryl, wherein        “heteroaryl” and “alkyl” have the meaning as defined further        above. Examples of heteroarylalkyl groups are heteroarylethyl        and benzyl, wherein benzyl is a particularly preferred        heteroarylalkyl group.    -   “Heteroarylalkyloxy” comprises the group —O-alkyl-heteroaryl,        wherein “heteroaryl” and “alkyl” have the meaning as defined        further above. Examples of heteroarylalkyloxy groups are        heteroarylethyloxy and benzyloxy.    -   “Heteroaryloxy” comprises the group-O-heteroaryl, wherein        heteroaryl” has the meaning as defined further above    -   “Heteroarylcarbonyl” is —C(O)-heteroaryl, wherein heteroaryl”        has the meaning as defined further above    -   “Heteroarylalkylcarbonyl” is —C(O)-alkyl-heteroaryl, wherein        “heteroaryl” and “alkyl” have the meaning as defined further        above    -   “Heteroarylalkyloxycarbonyl” is the group        —C(O)—O-alkyl-heteroaryl, wherein “heteroaryl” and “alkyl” have        the meaning as defined further above    -   “Heteroarylsulfonyl” is “SO₂-heteroaryl, wherein heteroaryl” has        the meaning as defined further above

“Heteroarylsulfonylamino” refers to the group —NH—SO₂-heteroaryl,wherein “heteroaryl” has the meaning as defined further above

-   -   “Heteroarylcarbonyl” refers to the group —CO-heteroaryl.    -   “Hydroxy” refers to the radical —OH.    -   “Nitro” refers to the radical-NO₂.    -   “Oxime” refers to the group —CH═N—OH.    -   “Phenyl” is the aromatic radical —C₆H₅. Whether a phenyl group        is substituted with one or more substituents, is specified        throughout this specification and the claims.    -   “Phenylalkyl” comprises the group -alkyl-phenyl, wherein        “phenyl” and “alkyl” have the meaning as defined further above.        Examples of phenylalkyl groups are phenylethyl and benzyl,        wherein benzyl is a particularly preferred phenylalkyl group.    -   “Phenylalkyloxy” comprises the group —O-alkyl-phenyl, wherein        “phenyl” and “alkyl” have the meaning as defined further above.        Examples of phenylalkyloxy groups are phenylethyloxy and        benzyloxy.    -   “Phenoxy” comprises the group-O-phenyl, wherein phenyl” has the        meaning as defined further above    -   “Phenylcarbonyl” is —C(O)-phenyl, wherein phenyl” has the        meaning as defined further above    -   “Phenylalkylcarbonyl” is —C(O)-alkyl-phenyl, wherein “phenyl”        and “alkyl” have the meaning as defined further above    -   “Phenylalkyloxycarbonyl” is the group —C(O)—O-alkyl-phenyl,        wherein “phenyl” and “alkyl” have the meaning as defined further        above    -   “Phenylsulfonyl” is —SO₂-phenyl, wherein phenyl” has the meaning        as defined further above    -   “Phenylsulfonylamino” refers to the group —NH—SO₂-phenyl,        wherein “phenyl” has the meaning as defined further above    -   “Sulfamoyl” includes the group —SO₂—NH₂.    -   “Sulfonylamino” includes the group —NH—SO₂H.    -   “Trifluormethyl” refers to the group —CF₃.

The term “GPR17 antagonists” as used herein includes compounds whichdecrease the GPR17 activity to 70% or less, 60% or less, 50% or less,preferably 40% or less, more preferably 30% or less, or even 20% or lessat the highest applied concentration compared to the GPR17 activityafter activation by RA 11-150; the term “GPR17 antagonist” includes fullantagonists, partial agonists, and inverse agonists, which may becompetitive or non-competitive in nature.

A preferred aspect of the invention relates to the method or assay ashereinbefore described, wherein in formula I

R1 is selected from the group consisting of methyl, CF₃, chloro, fluoro,bromo, iodo, phenyl and (C₅-C₆)heteroaryl, wherein the phenyl orheteroaryl group is optionally substituted with halogen, methyl ormethoxy;

R2 is selected from the group consisting of hydrogen, methyl, CF₃,chloro, fluoro, bromo, iodo and phenyl;

R3 is hydrogen, carboxymethyl, or carboxyethyl;

R4 and R5 are both hydrogen; and

n is 1.

In a particular preferred aspect, the GPR 17 agonist used in thescreening method and/or the assay of the present invention is selectedfrom the group comprising3-(2-carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dichloro-(1-carboxyethyl)-indole-2-carboxylicacid, 3-(2-carboxyethyl)-4,6-dimethyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-chloro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-difluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dibromo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-bromo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-iodo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-diiodo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-diphenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-phenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6,7-dichloro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-chloro-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-Bromo-2-carboxy-7-fluoro-1H-indole-2-carboxylicacid, 3-(2-carboxyethyl)-4,6-dimethoxy-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-phenoxyl H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-Benzyl-2-carboxy-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dihydroxy-1H-indole-2-carboxylic acid,3-(2-Carboxyethyl)-6-(4-fluorophenyl)-1H-indol-2-carboxylic acid,3-(2-carboxyethyl)-6-furanyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-thienyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-7-fluoro-6-phenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-(4-fluorophenyl)-7-fluoro-1H-indol-2-carboxylicacid, 3-(2-carboxyethyl)-6-furanyl-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-thienyl-7-fluoro-1H-indole-2-carboxylic acid, andsalts thereof.

In one preferred aspect, the biological activity of GPR17 is determinedin the method and assays of the present invention by measuring theincrease of ³⁵SGTP_(Y)S binding to the heterotrimeric G proteinsactivated by the receptor, the inhibition of cAMP formation and/or therelease of calcium from intracellular calcium stores, the increase ininositolphosphates (IP), and/or the recruitment of cytoplasmicβ-arrestin proteins, as well as the other methods described herein.

In one aspect, the GPR17 activity is determined in the methods andassays of the present invention by using (a) a transfected cell line,which is preferably selected from transfected CHO cells, astrocytomacells, COS7 cells or HEK293 cells, or membrane fractions thereof, or (b)native tissue, cells (and membrane fractions thereof), expressing GPR17to an extent sufficient to achieve functional activation. Examples ofnaturally GPR17 expressing cells are oligodendrocytic precursor cellswhich can be gained from newborn animals (P1, P2) as primary cellcultures. Hence, the term “contacting a test compound with GPR 17” orsimilar phrases used herein include the addition of test compounds orputative GPR17 antagonists to cell systems or cell fragments(over)expressing GPR17.

A variety of methods, means and signals can be used as “read outssystems” in the screening method of the present invention, and are allencompassed by the method of the present invention. Such read outsystems will be used to determine the activity of GPR17 or functionalfragments thereof after its activation by the agonists of the inventionand the response to test compounds, preferably the response toantagonists.

Accordingly, in one aspect the method and/or screening assay (e.g. inform of a kit) of the present invention further comprise means fordetermining the GPR17 activity (“read out system”). Said means fordetermining the GPR17 activity are preferentially selected from one ormore of

-   -   (a) means for determining the release of calcium from        intracellular calcium stores associated with GPR17 activation,        preferably a cell membrane permeable indicator, which binds to        calcium released in the cell thereby providing a measurable        signal, preferably fluorescence or luminescence,    -   (b) means for determining GTP_(Y)S binding to heterotrimeric G        proteins thereby proving a measurable signal, preferably        ³⁵SGTP_(Y)S, the radioactivity of which will be incorporated        into heterotrimeric G proteins,    -   (c) means for determining the inhibition of cAMP formation or        its elevation associated with GPR17 activation, preferably a        stimulator of the adenylyl cyclase (e.g. forskolin) and a        suitable cAMP indicator system,    -   (d) means for determining the increase in inositolphosphates        (IP), quantified as IP1, e.g. a suitable IP1 detector system    -   (e) means for determining the recruitment of cytoplasmic        β-arrestin proteins, preferably detection of β-arrestin        translocation to the receptor using fluorescence or        bioluminescence resonance energy transfer.

In one preferred aspect of the present invention, the read out systemused in the method and/or screening assay of the present invention isbased on the measurement of cAMP formation as (inverse) indicator ofGPR17 activity and includes forskolin as stimulator of the adenylylcyclase, and a competitive immunoassay as cAMP indicator system, whichpreferably provides a fluorescence signal. Such an indicator system isdescribed in more detail in Example 3 herein.

One preferred aspect of the present invention relates to the method orscreening assay of the present invention used for identifyingantagonists of GPR17.

One preferred aspect of the present invention relates to a method ofidentifying a GPR17 antagonist.

One preferred aspect of the present invention relates to a method ofmanufacturing a GPR17 modulator, preferably a GPR17 antagonistcomprising the step of determining the compound's ability to modulatethe biological activity of GPR17 by subjecting said compound to themethod and/or assay of the present invention. For example, a library ofnew compounds may be selected and screened for the compounds' ability tomodify the GPR 17 response by applying the method and/or assay of thepresent invention.

Hence, one aspect of the present invention relates to a method ofmanufacturing a GPR 17 modulator, comprising the steps of

-   -   (a) identifying a compound that modulates GPR17 activity by the        method described herein, and    -   (b) manufacturing a compound identified as GPR17 modulator.

One aspect of the present invention relates to a method of manufacturinga GPR17 antagonist comprising the following subsequent steps:

-   -   (1) selecting at least one compound which acts as a GPR 17        antagonist when tested in the method and/or the assay of the        present invention,    -   (2) selecting one or more analogs of the GPR 17 antagonist(s)        selected in step (1),    -   (3) subjecting the one or more GPR 17 antagonist analogs of        step (2) to the method and/or assay of the present invention        thereby determining the GPR 17 antagonistic properties of said        GPR 17 antagonist analogs,    -   (4) optionally repeating steps (1) to (3) one or more times by        producing further chemical analogs of the GPR 17 antagonist(s)        thereby identifying GPR17 antagonists with improved GPR17        antagonistic properties,    -   (5) selecting a compound for synthesis, and    -   (6) synthesizing or having synthesized the compound selected in        step (5).

One preferred aspect of the present invention relates to a method ofidentifying a GPR17 antagonist useful for the treatment, alleviation orprevention of multiple sclerosis comprising the screening method of thepresent invention and/or comprising a step of using the screening assayof the present invention.

One aspect of the invention is a method of treating, alleviating orpreventing a GPR17 mediated disease, such as spinal cord injury,multiple sclerosis, cerebral, cardiac and renal ischemia, and preferablymultiple sclerosis or an ischemic brain insult such as stroke in apatient in need thereof comprising the steps of

-   -   (a) identifying a GPR17 antagonist using the screening method        and/or the assay of the present invention,    -   (b) administering said identified GPR17 antagonist in a        therapeutically effective concentration, optionally together        with one or more pharmaceutically acceptable excipients, to said        patient in need thereof.

One aspect of the present disclosure relates to a GPR17 antagonist,which has been identified using the screening method or assay of thepresent invention, and the use of such GPR17 antagonist in therapy,particularly in the treatment, alleviation or prevention of a GPR17mediated disease, such as spinal cord injury, multiple sclerosis,cerebral, cardiac and renal ischemia, preferably of multiple sclerosisor stroke.

One aspect of the present invention relates to the use of the GPR17agonist of the present invention in a screening assay and/or in a methodwhich is suitable to identify GPR17 modulators, preferably GPR17antagonists. In one aspect said GPR17 agonists may be used to screen forGPR 17 antagonists among a multitude of test compounds for which theGPR17 affinity and/or functional activity at GPR17 are basicallyunknown. In one aspect of the present invention, said GPR17 agonists maybe used to determine (including confirming) the GPR 17 affinity orfunctional activity towards GPR17 of a single compound or a selectedgroup of compounds.

The present invention is further illustrated by the non-limitingexamples below.

EXPERIMENTAL SECTION Examples Example (1) hGPR17 Expressing Cell Systemsand Media

The sequence for the human GPR17 (short isoform) was subcloned into thevector pLXSN (Clontech Laboratories, CA 94043, USA), which can be usedfor retroviral transfection of the human astrocytoma cell line 1321N1.Since it was postulated by Ciana et. al. that GPR17 is activated byuracil nucleotides such as UDP, UDP-glucose and UDP-galactose, 1321N1astrocytoma cells were chosen as an appropriate test system, because itwas known that this cell line does not express endogenous nucleotidereceptors. For the retroviral transfection of 1321N1 astrocytoma cellsthe packaging cell line GP+envAM12 was cultivated in HXM medium (50 mlof foetal calf serum (FCS), 5 ml of penicillin G/streptomycin solution(final concentration 100 U/ml penicillin, 100 μg/ml streptomycin), 1%ultra glutamine, 0.75 ml of hypoxanthine (10 mg/ml), 12.5 ml of xanthine(10 mg/ml), 1.25 ml of mycophenolic acid (10 mg/ml) and 2 ml ofhygromycin B (50 mg/ml) were added to 500 ml DMEM). One day beforetransfection 1.5×10⁶ cells were seeded and incubated overnight at 37°C., 5% CO₂ and 95% humidity in a 25 cm² cell culture flask containingmedium without antibiotics. For at least two hours before transfectionthe medium was exchanged for 6.25 ml DMEM medium without any additive.Two solutions were prepared for the transfection. The first solutionconsisted of 25 μl of Lipofectamine™ 2000 (final concentration 2%) and600 μl of DMEM medium without any additives while the second solutionwas composed of 6.25 μg of pLXSN-GPR17 and 3.75 μg of pLXSN-VSV-G vectorDNA. The second solution was filled up with DMEM medium without anyadditives. Solution one was incubated for 5 min at room temperaturebefore both solutions were combined and incubated for 20 min, also atroom temperature. Afterwards the sample was added to the packaging cellline and incubated for 12-15 h. The medium was then replaced with 3 mlof fresh medium containing 30 μl of a 500 mM sodium butyrate solution.The virus production was stimulated by sodium butyrate and took place byincubation for 48 h at 32° C. and 95% humidity. One day before infection500,000 of 1321N1 astrocytoma cells were seeded in a 25 cm² cell cultureflask. After 48 h of virus production, the retroviruses were harvestedand sterilised by filtration through a filter with a pore size of 45 μm.The host cell medium was replaced with the sterilized virus supernatantcontaining 6 μl of polybrene solution (4 mg/ml) and the infection of thehost cells took place for further 48 h of incubation at 37° C., 95%humidity and 5% CO₂. After incubation, the transfected cells weretransferred to a 175 cm² cell culture flask and selected by addingmedium containing G418. The efficiency of infection and stabletransfection using this method was up to 95%.

In order to investigate potential differences in receptor signalling asa function of the cellular background, GPR17 was additionally expressedand analysed in a further cell system. Therefore, a recombinant Chinesehamster ovary (CHO) cell line stably expressing GPR17 was generatedusing the Flp-In™ T-Rex™ expression system. Here, expression of GPR17was induced after adding doxycycline.

Medium for Retrovirally Transfected 1321N1 Astrocytoma Cells

G418 (800 μg/ml G418) was added to DMEM medium with 10% FCS forpreparation of medium to cultivate 1321N1 astrocytoma cells stablyexpressing GPR17.

Medium for Flp-In T-Rex-CHO (FLIPR) Cells (=CHO-GPR17 Cells)

DMEM/F-12 medium with 10% FCS, 30 μg/ml of blasticidin and 500 μg/ml ofhygromycin B was used for culturing stable CHO cells generated by usingthe Flp-In T-rex system. Doxycycline was added up to a concentration of1 μg/ml for the induction of receptor expression in this cell line.

Example (2) Measurement of Ligand Activity by Calcium Mobilization Assay

PLC activation and IP₃ accumulation result in a release of calcium fromintracellular calcium stores, for example the endoplasmic reticulum. Inorder to detect the released calcium, cells are loaded with a cellmembrane permeable dye, which is converted within the cell into itsactive form by the cleavage of ester bonds. The active form of the dyebinds the released calcium. The fluorescence properties of the dyechange after calcium binds to the dye. These changes can be detectedautomatically and represent the ligand-dependent receptor activation.Cells from two confluently grown (80 to 90%) 175 cm² cell culture flaskswere needed for a calcium assay using the NOVOstar microplate reader(approximately 150,000 cells per well). Cells were harvested afterremoving the medium, washed once with PBS buffer and detached using atrypsin/EDTA solution. After incubation for 45 min at 37° C. and 5% CO₂,the cells were pelleted by centrifugation (5 min, 200 g). Cells wereresuspended in 994 μl of Krebs HEPES buffer (KHB; 118.6 mM NaCl, 4.7 mMKCl, 1.2 mM KH₂PO₄, 4.2 mM NaHCO₃, 11.7 mM D-glucose, 10 mM HEPES (freeacid), 1.3 mM MgSO₄ and 1.2 mM CaCl₂, pH 7.4) and loaded in the absenceof light with fluorescent, namely Oregon Green 488 BAPTA-1/AM dye, byincubating them for one hour at 28° C. and 700 rpm in an EppendorfThermomixer in the presence of Pluronic F-127. Pluronic F-127 wassupplemented so that the cells could better absorb the dye. The cellswere washed twice with 1 ml of KHB buffer (three centrifugation steps,each for 15 sec at 2700 rpm) before being seeded in a 96-well microplatein a total volume of 180 μl. A 10-fold concentrated agonist solution (35μl) was added to another 96-well microplate with a V-profile. Bothplates were placed into the NOVOstar microplate reader and incubatedthere for 20 min. After incubation, the required gain was determined bythe microplate so that the fluorescence background of the cells wasbetween 38,000 and 42,000 fluorescence units. This background is anoptimal range for starting the measurement, because it is in the middleof the reader's detection range. Normally, the background in each wellof a microplate should be the same. The background was determined byperforming a so-called validation. A validation is performed to detectirregularities in cell densities between different wells, fluorescenceor absorption properties as well as unspecific antagonist effects. Theseirregularities are noticeable when the background is significantlyincreased or decreased compared to the normal range. Afterwards, 20 μlof agonist were injected from the source plate to the measurement plateby the injector unit of the NOVOstar, and the increase in fluorescencewas determined as a function of the calcium efflux resulting fromreceptor stimulation.

Example (3) Inhibition of Forskolin-Stimulated Intracellular cAMPAccumulation by Ligand

Two signal transduction pathways that influence the activity of theadenylyl cyclase in oppositional ways can be activated upon receptoractivation. Whereas the Gs pathway stimulates the adenylylcyclase-dependent catalysation of cAMP formation, the Gi pathwayinhibits it. The detection of cAMP accumulation is based on the HTRF®technology described above. In this case a monoclonal antibody specificto cAMP was the donor fluorophore while the acceptor fluorophore d2 wasfused to cAMP. The analysis of the Gi pathway was based on the directactivation of the adenylyl cyclase, for example, by forskolin. Uponactivation, the Gi pathway inhibits the forskolin-dependent activationof the adenylyl cyclase, leading to decreased cAMP accumulation. Theinhibition of forskolin-stimulated cAMP accumulation in 1321N1 or CHOcells was performed using the HTRF® cAMP dynamic kit (Cisbio Bioassays,BP 84175, France). Cells were resuspended in assay buffer with 1 mM3-isobutyl-1-methylxanthine (IBMX) supplemented and dispensed in384-well microplates at a density of 50,000 cells/well in a volume of 5μl. After preincubation in assay buffer for 30 min, the cells werestimulated by adding 5 μl of agonist in the respective concentration offorskolin. The final concentration of forskolin was dependent on thecell line which was employed in the assay. Adenylyl cyclase wasstimulated with 1 μM forskolin in 1321N1-GPR17 cells and with 10 μMforskolin in CHO-GPR17 cells, followed by incubation for 30 min at roomtemperature. The reaction was terminated by lysis of the cells, whichresults from the addition of 5 μl of d2-conjugate followed by theaddition of 5 μl of anti-IP1-cryptate (both supplements are diluted inconjugate and lysis buffer). The assay was incubated for 60 min at roomtemperature and time-resolved FRET signals were measured at anexcitation wavelength of 320 nm using the Mithras LB 940 multimodereader (Berthold technologies, D-75323 Bad Wildbad). The evaluation ofHTRFR data was performed following the instructions of the kitmanufacturer. Data analysis was based on the fluorescence ratio emittedby labeled cAMP (665 nm) over the light emitted by the europiumcryptate-labelled anti-cAMP (620 nm). Levels of cAMP were normalised tothe amount of cAMP elevated by 10 μM or 1 μM forskolin alone.

Example (4) Description of ³⁵SGTP_(y)S Binding Assay

[³⁵S]GTP_(γ)S binding experiments were carried out in 96-well platesusing 5 μg of protein/well. Membranes were added to 400 μl of incubationbuffer (10 mM HEPES, 10 mM MgCl₂, 100 mM NaCl, pH 7.4) containing 10 μMGDP and RA-II-150 in the indicated concentrations. Reactions werestarted by adding 50 μl of 0.07 nM [³⁵S]GTP_(γ)S and then incubated for60 min at 30° C. Membrane-bound radioactivity was separated by vacuumfiltration. After drying, glass fiber filter mats were melted withscintillation wax, and radioactivity was measured by liquidscintillation counting.

Example 5 β-Arrestin Assay

Arrestin recruitment was detected in BRET² assays using HEK293 cellsstably expressing human GPR17-Rluc and GFP2-β-arrestin2.

Cells were detached by trypsinization, counted and washed once in assaybuffer (HBSS, 20 mM HEPES (S12), pH 7.0). After centrifugation at 800rpm for 4 min, the pelleted cells were resuspended in an appropriatevolume of assay buffer to a density of 1×10⁶ cells per ml. In order tostabilize readings, cells were allowed to incubate at 28° C. for 30 minwhile slowly shaking (180 rpm), prior to experiments.

The coelenterazine 400A stock solution was freshly diluted (3:100) inPBS containing 20% of ethanol to obtain a 30 μM solution, which wasalways kept in the dark, due to its light-sensitivity.

As a first step, the agonist solutions were prepared and 10 μl of each8-fold dilution was dispensed in the 384-well assay plate. DMSOconcentrations were adjusted and did not exceed 0.1% in finalconcentrations.

Harvested and stabilized cells were manually distributed (70 μl, 70.000c/well) to the assay plate containing the agonist dilutions. Cells werestimulated at 28° C. at 450 rpm for 5 min. Following cell stimulation,Coelenterazine 400A (C10, 10 μl) was injected by the Mithras injector 3at a final concentration of 3.3 μM. Two seconds after the injections,the light output from the well was measured at 400 and 515 nm by use ofMithras LB 940 plate reader, which allows the sequential integration oflight signals detected with two filter settings. The BRET signal,milliBRET ratio, was calculated by the quotient of the fluorescenceemitted by GFP2-β-arrestin2 (515 nm) over the light emitted by theGPR17-Rluc (400 nm).

Example 6 Inositolphosphate IP1 Assay

Agonist induced IP1 accumulation was determined using the HTRF kit(Cisbio, France) according to manufacturer's instructions. On the day ofexperiment the required amount of cells were harvested and washed twicein HBSS (20 mM HEPES), including two centrifugation steps for 15 s at2700 rpm. Pelleted cells were resuspended in appropriate volume ofstimulation buffer (containing LiCl) and dispensed at 100.000 cells/7μl/well in 384-well microtiter plates. Following incubation at 37° C.for 30 min, cells were stimulated by addition of 7 μl 2-foldconcentrated agonists diluted in stimulation buffer. If the impact ofantagonists was investigated, they were added 30 min prior to agonistaddition and incubated at 37° C. After another 30 min incubation at 37°C., the reaction was terminated by addition of 3 μl of IP-d2 followed by3 μl of europium(Eu)-cryptate-labeled IP1 Mab diluted (1:20) in lysisbuffer. The plates were allowed to incubate for 60 min at roomtemperature and were then read by Mithras LB940 (Berthold) with 100 μsdelay and 200 μs window time.

Example 7 GPR17 Antagonist Screening Assay to Identify GPR17 Antagonists

GPR17-expressing cells were pre-incubated with the test compounds for adefined period of time (for example 5-30 minutes, longer and shorterincubations are possible) followed by addition of agonist at a fixedconcentration, for example its EC₈₀, which is the concentration ofagonist producing 80% of its maximal response. Under these conditions,inhibitors of GPR17 function display concentration-dependent inhibitionof the agonist signals as exemplified in FIG. 6 with a GPR17 inhibitor.

Example 8 Synthesis of GPR 17 Agonists

A. General Procedure for the Japp-Klingemann Reaction to Form IndoleDiethyl Esters.

Aniline (1 eq) was dissolved in concentrated HCl (3 eq) diluted withwater (˜2 mL/mmol of aniline), and cooled to 0° C. Sodium nitrite (2.5M, 1 eq) in water was added dropwise such that the temperature remainedbelow 5° C. Sodium acetate (4.5 M, 5.5 eq) in water was added to thesolution followed by ethyl 2-oxocyclopentanecarboxylate (1 eq). Thereaction was stirred at 0° C. for 15 min and then allowed to warm toroom temperature over a period of 1 hour. The aqueous solution wasextracted with chloroform (˜100 mL/mmol of aniline). The organic layerwas dried over sodium sulphate, filtered and evaporated under reducedpressure. To the dark oily mixture was added a solution of sodiumcarbonate (−0.7 M aq., 1.1 eq) and stirred under reflux for 10 min. Themixture was allowed to cool to room temperature and carefully acidifiedwith 6 N HCl. The precipitate was extracted with chloroform, dried oversodium sulphate, filtered and evaporated under reduced pressure. Theresidue was the dissolved in ethanol (1 mL/mmol of aniline) containingconcentrated sulphuric acid (−0.1 mL) and refluxed overnight. Thereaction was cooled to room temperature and the solvent concentratedunder vacuum. The crude sample was purified by column chromatographyusing ethyl acetate/hexane 1/4. The desired compound was recrystallizedfrom hot hexane.

B. General Procedure for the Alkylation of the Indole Diester Analogues.

A mixture of indole diester (1 eq), potassium carbonate (1 eq) andalkylating agent (3 eq) in the appropriate solvent was refluxedovernight. The solvent was evaporated under reduced pressure and themixture extracted with a mixture of ethyl acetate/saturated NaHCO₃solution. The organic layer was dried over sodium sulphate, filtered andconcentrated under vacuum. The resulting crude sample was purified bycolumn chromatography.

C. General Procedure for the Saponification of Indole Diethyl Esters.

The diester was dissolved in THF (10 mL/mmol of indole diethyl ester)and diluted with an equal volume of water and then treated with LiOH.H₂O(10 eq). The reaction mixture was stirred at room temperature overnight,diluted with water, acidified, and extracted with ethyl acetate. Theorganic layer was dried over sodium sulphate, filtered and concentratedunder reduced pressure. The product was recrystallized from hot ethylacetate/hexane.

D. Synthesis of Example Compounds

(a) 3-(2-Carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid

Reference, Incorporated Herein by Reference in its Entirety:

-   Salituro, Francesco G.; Harrison, Boyd L.; Baron, Bruce M.; Nyce,    Philip L.; Stewart, Kenneth T.; Kehne, John H.; White, H. Steven;    McDonald, Ian A. 3-(2-Carboxy1H-indole-2-carboxylic acid-based    antagonists of the NMDA (N-methyl-D-aspartic acid) receptor    associated glycine binding site. Journal of Medicinal Chemistry    (1992), 35, 1791-9.

(b) 3-(2-Carboxyethyl)-4,6-dichloro-(1-carboxyethyl)-indole-2-carboxylicacid (i)4,6-Dichloro-1,3-bis-(2-ethoxycarbonylethyl)-1H-indole-2-carboxylic acidethyl ester

The desired compound was obtained using ethyl 3-bromopropionate asalkylating agent and acetonitrile as reaction solvent, andrecrystallized from hot methanol (yield 77%). ¹H NMR (500 MHz, CDCl₃) δ1.2 (t, J=7.25 Hz, 3H), 1.23 (t, J=7.25 Hz, 3H), 1.41 (t, J=7.25 Hz,3H), 2.64 (m, 2H), 2.77 (t, J=7.25 Hz, 2H), 3.59 (m, 2H), 4.09 (q,J=7.25 Hz, 2H), 4.12 (q, J=7.25 Hz, 2H), 4.41 (q, J=7.25 Hz, 2H), 4.67(t, J=7.25 Hz, 2H), 7.1 (d, J=1.55 Hz, 1H), 7.31 (d, J=1.6 Hz, 1H) ppm.

(ii) 1,3-Bis-(2-carboxyethyl)-4,6-dichloro-1H-indole-2-carboxylic acid

(Yield 50%); mp 229° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 2.49 (m, 2H), 2.65(t, J=7.25 Hz, 2H), 3.44 (t, J=8.1 Hz, 2H), 4.65 (t, J=7.25 Hz, 2H), 7.2(d, J=1.6 Hz, 1H), 7.76 (d, J=1.55, 1H), 12.69 (br, 3H) ppm; ¹³C NMR(500 MHz, DMSO-d₆) 20.63, 34.74, 36.31, 110.47, 121.18, 127.19, 128.75,129.03, 138.64, 162.92, 172.35, 173.66. EIMS (m/z, %) 374.0 (M⁺, 100).

(c) 3-(2-Carboxyethyl)-4,6-dimethyl-1H-indole-2-carboxylic acid

Reference Incorporated Herein by Reference in its Entirety:

-   Takahashi, Kenji; Kasai, Masayasu; Ohta, Masaru; Shoji, Yoshimichi;    Kunishiro, Kazuyoshi; Kanda, Mamoru; Kurahashi, Kazuyoshi;    Shirahase, Hiroaki Novel Indoline-Based Acyl-CoA:Cholesterol    Acyltransferase Inhibitor with Antiperoxidative Activity:    Improvement of Physicochemical Properties and Biological Activities    by Introduction of Carboxylic Acid. Journal of Medicinal Chemistry    (2008), 51, 4823-4833.

(d) 3-(2-Carboxyethyl)-6-chloro-1H-indole-2-carboxylic acid

Reference:

-   Salituro, Francesco G.; Harrison, Boyd L.; Baron, Bruce M.; Nyce,    Philip L.; Stewart, Kenneth T.; Kehne, John H.; White, H. Steven;    McDonald, Ian A. 3-(2-Carboxy1H-indole-2-carboxylic acid-based    antagonists of the NMDA (N-methyl-D-aspartic acid) receptor    associated glycine binding site. Journal of Medicinal Chemistry    (1992), 35, 1791-9.

(e) 3-(2-Carboxyethyl)-4,6-difluoro-1H-indole-2-carboxylic acid

Reference:

-   Salituro, Francesco G.; Harrison, Boyd L.; Baron, Bruce M.; Nyce,    Philip L.; Stewart, Kenneth T.; Kehne, John H.; White, H. Steven;    McDonald, Ian A. 3-(2-Carboxy1H-indole-2-carboxylic acid-based    antagonists of the NMDA (N-methyl-D-aspartic acid) receptor    associated glycine binding site. Journal of Medicinal Chemistry    (1992), 35, 1791-9.

(f) 3-(2-Carboxyethyl)-4,6-dibromo-1H-indole-2-carboxylic acid (i)3,5-Dibromobenzenediazonium salt (Mixture I)

To a well stirred suspension of 3,5-dibromoaniline (2.51 g, 10 mmol) in16.6 ml HCl (5 M) at 0° C. was dropwise added a solution of sodiumnitrite 1.38 g (20 mmol, 2 equiv) in 8 ml water, previously cooled to 0°C. The addition of sodium nitrite solution was slow, in order to keepthe temperature of the mixture below 8° C. The resulting orange-redmixture was stirred at 0° C. for further 20 min.

(ii) 2-(Ethoxycarbonyl)cyclopentanone (mixture II)

2-(Ethoxycarbonyl)cyclopentanone (2.512 ml, 1.344 g, 15 mmol) wasdissolved in ethanol (4.2 ml) and cooled to 0° C. Then, a potassiumhydroxide solution (5.040 g, 90 mmol, 6 equiv.) in water (5 ml)previously cooled to 0° C. was added dropwise within ca. 30 min in orderto keep the temperature below 8° C. The mixture turned to a white-milkycolor, and the final mixture was stirred at 0° C. for further 30 min.

(iii)(E/Z)-5-(2-(3,5-Dibromophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoic acid

Ice (50 g) was added to mixture II with stirring at 0° C., followed bythe addition of mixture I, and stirring continued for 1 h at 40° C. Thecombined mixtures were then let to cool to rt and the pH wassubsequently adjusted to 4-5 by adding 1 M HCl. The desired product wasextracted with diethyl ether (3×50 ml). The organic layer was collected,dried over magnesium sulfate, filtered, and the filtrate was evaporatedto dryness yielding gummy material (95-99%). This material was usedwithout further purification for the next step.

(iv) (E/Z)-Diethyl 2-(2-(3,5-dibromophenyl)hydrazinyl)hex-2-enedioate

(E/Z)-5-(2-(3,5-Dibromophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoic acid(4.361 g, 10 mmol) was dissolved in absolute ethanol (100 ml) followedby the addition of conc. sulfuric acid (2.7 ml, 50.5 mmol, 5.1 equiv.).The mixture was then allowed to reflux for 1 h at 100° C. Then theethanol was evaporated and the residue was treated with 100 ml ofice-water. The aqueous solution was extracted with dichloromethane (3×50ml); the organic layer was dried over magnesium sulfate, filtered andconcentrated. The residue was purified by silica gel columnchromatography using 20% of ethyl acetate in cyclohexane yielding awhite solid in 85% yield.

(v) Ethyl 4,6-dibromo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate

A mixture of p-toluenesulfonic acid (2.954 g, 15 mmol, 1.5 equiv.) and100 ml of dry toluene was refluxed for 1 h at 140° C.; water wascontinuously removed by means of a Dean-Stark trap. Subsequently, 4.64 g(10 mmol) of the starting material ((E/Z)-diethyl2-(2-(3,5-dibromophenyl)hydrazinyl)hex-2-enedioate) dissolved in aminimum amount of dry toluene (ca. 15 ml) was added and the mixture wasrefluxed for 5 h. Then it was allowed to cool down to rt, and toluenewas removed under reduced pressure and the residue was dissolved indichloromethane and washed with water. The organic layer was dried overmagnesium sulfate, filtered, and evaporated to dryness. The residue waspurified by silica gel column chromatography using 20% of ethylacetate/cyclohexane as eluent to yield a light-beige-colored solid in75%. ¹H-NMR (DMSO-d₆): δ 1.15, 1.34 (each t, 3H, ³J=7.1 Hz, CH₃); 2.55(t, 2H, ⁴J=8.2 Hz, 2′-H); 3.54 (t, 2H, ⁴J=8.2 Hz, 1′-H); 4.05, 4.35(each q, 2H, J=7.1 Hz, CH₂); 7.42 (d, 1H, ⁴J=1 Hz, 5-H); 7.60 (d, 1H,⁴J=1 Hz, 7-H); 12.06 (s, 1H, NH). ¹³C-NMR (DMSO-d₆): δ 14.17, 14.20(2CH₃, each C═OOCH₂CH₃); 19.9 (C-2′); 36.0 (C-1′); 59.9, 60.9 (2CH₂,each C═OOCH₂CH₃); 114.9 (C-7); 115.5 (C-4); 117.1 (C-6); 121.6 (C-3);123.5 (C-2); 125.7 (C-3a); 126.4 (C-5); 138.0 (C-7a); 161.1 (2′-CO₂Et);171.9 (2-CO₂Et). LC-MS (m/z): 465 [M+NH₄ ⁺]⁺, 448 [M]⁺, 446 [M]⁻. Purity(LC-MS): 98%.

(vi) 3-(2-Carboxyethyl)-4,6-dibromo-1H-indole-2-carboxylic acid

Ethyl 4,6-dibromo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate (4.47g, 10 mmol) was dissolved in 25 ml tetrahydrofurane (THF) with stirringat rt. Then a solution of 1.26 g of lithium hydroxide trihydrate (3equiv.) in 25 ml water was added and the resulting mixture was let tostir at rt for 24 h. After completion of the reaction THF was removedunder reduced pressure and the pH was adjusted to 4-5, and the productwas extracted with diethyl ether (3×30 ml). The organic layers weredried over magnesium sulfate, filtered, and evaporated to dryness toyield a light-beige-colored solid in 95-100% yield. mp. 259° C.; wellsoluble in methanol, ethanol and ethyl acetate; slightly soluble inpetrol ether, water, hexane. ¹H-NMR (MeOH-d₄): δ 2.67 (t, 2H, ³J=8.4 Hz,CH₂); 3.71 (t, 2H, ³J=8.4 Hz, CH₂); 7.42 (d, 1H, ⁴J=1.6 Hz, C7-H); 7.62(d, 1H, ⁴J=1.6 Hz, C5-H). ¹³C-NMR (MeOH-d₄): δ 21.4 (C-2′); 37.9 (C-1′);115.9 (C-7); 117.0 (C-4); 118.8 (C-6); 123.9 (C-3); 125.5 (C-2); 127.9(C-3a); 128.2 (C-5); 139.6 (C-7a); 164.6 (2′-CO₂H); 170.1 (2-CO₂H).LC-MS (m/z): 392.0 [M]⁺; 390.0 [M]⁻. Purity (LC-MS): 99%.

(g) 3-(2-Carboxyethyl)-6-bromo-1H-indole-2-carboxylic acid (i)3-Bromobenzenediazonium salt (mixture I)

To a well stirred suspension of 3-bromoaniline (1.72 g, 10 mmol) in 16.6ml HCl (5 M) at 0° C. was dropwise added a solution of sodium nitrite1.38 g (20 mmol, 2 equiv) in 8 ml water, previously cooled to 0° C. Theaddition of sodium nitrite solution was slow, in order to keep thetemperature of the mixture below 8° C. The resulting orange-red mixturewas stirred at 0° C. for further 20 min.

(ii) 2-(Ethoxycarbonyl)cyclopentanone (mixture II)

2-(Ethoxycarbonyl)cyclopentanone (2.51 ml, 1.34 g, 15 mmol) wasdissolved in ethanol (4.2 ml) and cooled to 0° C. Then, a potassiumhydroxide solution (5.04 g, 90 mmol, 9 equiv.) in water (5 ml)previously cooled to 0° C. was added dropwise within ca. 30 min in orderto keep the temperature below 8° C. The mixture turned milky-white, andthe final mixture was stirred at 0° C. for further 30 min.

(iii) (E/Z)-5-(2-(3-Bromophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoicacid

Ice (50 g) was added to mixture II with stirring at 0° C., followed bythe addition of mixture I, and stirring continued for 1 h at 40° C. Thecombined mixtures were then let to cool to rt and the pH wassubsequently adjusted to 4-5 by adding 1 M HCl. The desired product wasextracted with diethyl ether (3×50 ml). The organic layer was collected,dried over magnesium sulfate, filtered, and the filtrate was evaporatedto dryness yielding gummy material (95-99%). This material was usedwithout further purification for the next step.

(iv) (E/Z)-Diethyl 2-(2-(3-bromophenyl)hydrazinyl)hex-2-enedioate

(E/Z)-5-(2-(3-bromophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoic acid(3.57 g, 10 mmol) was dissolved in absolute ethanol (100 ml) followed bythe addition of conc. sulfuric acid (2.7 ml, 50.5 mmol, 5.1 equiv.). Themixture was then allowed to reflux for 3 h at 100° C. Then the ethanolwas evaporated and the residue was treated with 100 ml of ice-water. Theaqueous solution was extracted with dichloromethane (3×50 ml); theorganic layer was dried over magnesium sulfate, filtered andconcentrated. The residue was purified by silica gel columnchromatography using 20% of ethyl acetate in cyclohexane yielding awhite solid in 85% yield.

(v) Ethyl 6-bromo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate

A mixture of p-toluenesulfonic acid (0.60 g, 3.12 mmol, 1.5 equiv.) and100 ml of dry toluene was refluxed for 1 h at 140° C.; water wascontinuously removed by means of a Dean-Stark trap. Subsequently, 0.80 g(2.1 mmol) of the starting material ((E/Z)-diethyl2-(2-(3-bromophenyl)hydrazinyl)hex-2-enedioate) dissolved in a minimumamount of dry toluene (ca. 15 ml) was added and the mixture was refluxedfor 2 h. Then it was allowed to cool down to rt, and toluene was removedunder reduced pressure and the residue was dissolved in dichloromethaneand washed with water. The organic layer was dried over magnesiumsulfate, filtered, and evaporated to dryness. The residue was purifiedby silica gel column chromatography using 20% of ethylacetate/cyclohexane as eluent to yield a light-beige-colored solid in49.3%. ¹H-NMR (DMSO-d₆) δ 1.09, 1.34 (each t, 3H, ³J=7.1 Hz, CH₃); 2.57(m, 2H, 2′-H); 3.27 (m, 2H, 1′-H); 3.98, 4.34 (each q, 2H, ⁴J=7.1 Hz,CH₂); 7.18 (dd, 1H, J=1.8 Hz, 5-H); 7.56 (dd, 1H, J=1.8 Hz, 7-H); 7.64(dd, 1H, J=8.5 Hz, 4-H); 11.69 (s, 1H, NH). ¹³C-NMR (DMSO-d₆) δ 14.10,14.25 (2CH₃, each C═OOCH₂CH₃); 19.9 (C-2′); 35.0 (C-1′); 59.9, 60.6(2CH₂, each C═OOCH₂CH₃); 114.9 (C-7), 117.9 (C-4), 121.7 (C-3), 122.5(C-6), 122.7 (C-5), 124.2 (C-2) 126.0 (C-3a) 136.9 (C-7a), 161.39(2′-CO₂Et), 172.29 (2-CO₂Et). LC-MS (m/z): 387 [M-NH4⁺]⁺, 370 [M]⁺, 368[M]⁻. Purity (LC-MS): 97%.

(vi) 3-(2-Carboxyethyl)-6-bromo-1H-indole-2-carboxylic acid

Ethyl 6-bromo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate (3.68 g,10 mmol) was dissolved in 25 ml tetrahydrofurane (THF) with stirring atrt. Then a solution of 1.26 g of lithium hydroxide trihydrate (3 equiv.)in 25 ml water was added and the resulting mixture was let to stir at rtfor 24 h. After completion of the reaction THF was removed under reducedpressure and the pH was adjusted to 4-5, and the product was extractedwith diethyl ether (3×30 ml). The organic layers were dried overmagnesium sulfate, filtered, and evaporated to dryness to yield alight-beige-colored solid in 95-100% yield. mp. 234-235° C. ¹H-NMR(DMSO-d₆) δ 2.50 (m, 2H, 2′-H); 3.23 (m, 2H, 1′-H); 7.16 (dd, 1H, J=1.8Hz, 5-H); 7.54 (dd, 1H, J=1.8 Hz, 7-H); 7.64 (dd, 1H, J=8.5 Hz, 4-H);11.57 (s, 1H, NH); 12.54 (b, 1H, 2CO₂H). ¹³C-NMR (DMSO-d₆) δ 19.9(C-2′); 35.2 (C-1′); 114.9 (C-7); 117.6 (C-4); 121.6 (C-3); 122.5 (C-6,C-5); 125.0 (C-2); 126.2 (C-3a); 136.8 (C-7a); 163.0 (2′-CO₂H); 174.0(2-CO₂H). LC-MS (m/z): 329 [M-NH4⁺]⁺, 312 [M]⁺, 312 [M]⁻. Purity(LC-MS): 99%.

(h) 3-(2-Carboxyethyl)-6-iodo-1H-indole-2-carboxylic acid (i)3-Iodobenzenediazonium salt (mixture I)

To a well stirred suspension of 3-iodoaniline (2.19 g, 10 mmol) in 16.6ml HCl (5 M) at 0° C. was dropwise added a solution of sodium nitrite1.38 g (20 mmol, 2 equiv.) in 8 ml water, previously cooled to 0° C. Theaddition of sodium nitrite solution was slow, in order to keep thetemperature of the mixture below 8° C. The resulting orange-red mixturewas stirred at 0° C. for further 20 min.

(ii) 2-(Ethoxycarbonyl)cyclopentanone (mixture II)

2-(Ethoxycarbonyl)cyclopentanone (2.51 ml, 1.34 g, 15 mmol) wasdissolved in ethanol (4.2 ml) and cooled to 0° C. Then, a potassiumhydroxide solution (5.04 g, 90 mmol, 9 equiv.) in water (5 ml)previously cooled to 0° C. was added dropwise within ca. 30 min in orderto keep the temperature below 8° C. The mixture turned to a white-milkycolor, and the final mixture was stirred at 0° C. for further 30 min.

(iii) (E/Z)-5-(2-(3-Iodophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoicacid

Ice (50 g) was added to mixture II with stirring at 0° C., followed bythe addition of mixture I, and stirring continued for 1 h at 40° C. Thecombined mixtures were then let to cool to rt and the pH wassubsequently adjusted to 4-5 by adding 1 M HCl. The desired product wasextracted with diethyl ether (3×50 ml). The organic layer was collected,dried over magnesium sulfate, filtered, and the filtrate was evaporatedto dryness yielding gummy material (95-99%). This material was usedwithout further purification for the next step.

(iv) (E/Z)-Diethyl 2-(2-(3-iodophenyl)hydrazinyl)hex-2-enedioate

(E/Z)-5-(2-(3-Iodophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoic acid(2.02 g, 5 mmol) was dissolved in absolute ethanol (100 ml) followed bythe addition of conc. sulfuric acid (1.35 ml, 25.5 mmol, 5.1 equiv.).The mixture was then allowed to reflux for 2 h at 100° C. Then theethanol was evaporated and the residue was treated with 100 ml ofice-water. The aqueous solution was extracted with dichloromethane (3×50ml); the organic layer was dried over magnesium sulfate, filtered andconcentrated. The residue was purified by silica gel columnchromatography using 20% of ethyl acetate in cyclohexane yielding awhite solid in 98% yield.

(v) Ethyl 6-iodo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate

A mixture of p-toluenesulfonic acid (2.85 g, 15 mmol, 1.5 equiv.) and100 ml of dry toluene was refluxed for 1 h at 140° C.; water wascontinuously removed by means of a Dean-Stark trap. Subsequently, 4.32 g(10 mmol) of the starting material ((E/Z)-diethyl2-(2-(3-iodophenyl)hydrazinyl)hex-2-enedioate) dissolved in a minimumamount of dry toluene (ca. 15 ml) was added and the mixture was refluxedfor 1 h. Then it was allowed to cool down to rt, and toluene was removedunder reduced pressure and the residue was dissolved in dichloromethaneand washed with water. The organic layer was dried over magnesiumsulfate, filtered, and evaporated to dryness. The residue was purifiedby silica gel column chromatography using 20% of ethylacetate/cyclohexane as eluent to yield a light-beige-colored solid in61.3%. ¹H-NMR (DMSO-d₆) δ 1.09, 1.34 (each t, 3H, ³J=7 Hz, CH₃); 2.56(m, 2H, 2′-H); 3.25 (m, 2H, 1′-H), 3.98, 4.34 (each q, 2H, ⁴J=7 Hz,CH₂); 7.33 (dd, 1H, J=1.8 Hz, 5-H); 7.50 (d, 1H, J=8.5 Hz, 4-H); 7.77(d, 1H, J=1.8 Hz, 7-H); 11.66 (s, 1H, NH). ¹³C-NMR (DMSO-d₆) δ 14.1,14.3 (2CH₃, each C═OOCH₂CH₃); 19.9 (C-2′), 34.95 (C-1′); 59.9, 60.6(2CH₂, each C═OOCH₂CH₃); 90.03 (C-6), 121.0 (C-7); 121.7 (C-3); 122.6(C-4); 123.7 (C-2); 126.3 (C-3a); 128.1 (C5); 137.5 (C-7a); 161.4(2′-CO₂Et), 172.3 (2-CO₂Et). LC-MS (m/z): 433 [M-NH4⁺]⁺, 416 [M]⁺, 414[M]⁻. Purity (LC-MS): 98%.

(vi) 3-(2-Carboxyethyl)-6-iodo-1H-indole-2-carboxylic acid

Ethyl 6-iodo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate (4.15 g,10 mmol) was dissolved in 25 ml tetrahydrofurane (THF) with stirring atrt. Then a solution of 1.26 g of lithium hydroxide trihydrate (3 equiv.)in 25 ml water was added and the resulting mixture was let to stir at rtfor 24 h. After completion of the reaction THF was removed under reducedpressure and the pH was adjusted to 4-5, and the product was extractedwith diethyl ether (3×30 ml). The organic layers were dried overmagnesium sulfate, filtered, and evaporated to dryness to yield alight-beige-colored solid in 95-100% yield. mp. 230-232° C. ¹H-NMR(DMSO-d₆) δ 2.49 (m, 2H, 2′-H); 3.22 (m, 2H, 1′-H); 7.31 (dd, 1H, J=1.8Hz, 5-H); 7.50 (d, 1H, J=8.5 Hz, 4-H); 7.74 (d, 1H, J=1.8 Hz, 7-H);11.52 (s, 1H, NH); 12.53 (b, 2H, 2CO₂H). ¹³C-NMR (DMSO-d₆) δ 19.9 (C2′);35.2 (C1′); 89.6 (C-6); 121.0 (C-7); 121.6 (C-3); 122.7 (C-4); 124.6(C-2); 126.5 (C-3a); 127.9 (C-5); 137.3 (C-7a); 163.0 (2′-CO₂H); 174.0(2-CO₂H). LC-MS (m/z): 377 [M-NH4⁺]⁺, 360 [M]⁺, 358 [M]⁻. Purity(LC-MS): 98%.

(I) 3-(2-Carboxyethyl)-4,6-diiodo-1H-indole-2-carboxylic acid (i)3,5-Diiodobenzenediazonium salt (mixture I)

To a well stirred suspension of 3,5-diiodoaniline (3.45 g, 10 mmol) in16.6 ml HCl (5 M) at 0° C. was dropwise added a solution of sodiumnitrite 1.38 g (20 mmol, 2 equiv.) in 8 ml water, previously cooled to0° C. The addition of sodium nitrite solution was slow, in order to keepthe temperature of the mixture below 8° C. The resulting orange-redmixture was stirred at 0° C. for further 20 min.

(ii) 2-(Ethoxycarbonyl)cyclopentanone (mixture II)

2-(Ethoxycarbonyl)cyclopentanone (2.51 ml, 1.34 g, 15 mmol) wasdissolved in ethanol (4.2 ml) and cooled to 0° C. Then, a potassiumhydroxide solution (5.04 g, 90 mmol, 6 equiv.) in water (5 ml)previously cooled to 0° C. was added dropwise within ca. 30 min in orderto keep the temperature below 8° C. The mixture turned to a white-milkycolor, and the final mixture was stirred at 0° C. for further 30 min.

(iii) (E/Z)-5-(2-(3,5-Diiodophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoicacid

Ice (50 g) was added to mixture II with stirring at 0° C., followed bythe addition of mixture I, and stirring continued for 1 h at 40° C. Thecombined mixtures were then let to cool to rt and the pH wassubsequently adjusted to 4-5 by adding 1 M HCl. The desired product wasextracted with diethyl ether (3×50 ml). The organic layer was collected,dried over magnesium sulfate, filtered, and the filtrate was evaporatedto dryness yielding gummy material (95-99%). This material was usedwithout further purification for the next step.

(iv) (E/Z)-Diethyl 2-(2-(3,5-diiodophenyl)hydrazinyl)hex-2-enedioate

(E/Z)-5-(2-(3,5-Diiodophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoic acid(5.30 g, 10 mmol) was dissolved in absolute ethanol (100 ml) followed bythe addition of conc. sulfuric acid (2.7 ml, 50.5 mmol, 5.1 equiv.). Themixture was then allowed to reflux for 1 h at 100° C. Then the ethanolwas evaporated and the residue was treated with 100 ml of ice-water. Theaqueous solution was extracted with dichloromethane (3×50 ml); theorganic layer was dried over magnesium sulfate, filtered andconcentrated. The residue was purified by silica gel columnchromatography using 20% of ethyl acetate in cyclohexane yielding awhite solid in 85% yield.

(v) Ethyl 4,6-diiodo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate

A mixture of p-toluenesulfonic acid (2.43 g, 12.75 mmol, 1.5 equiv.) and100 ml of dry toluene was refluxed for 1 h at 140° C.; water wascontinuously removed by means of a Dean-Stark trap. Subsequently, 4.75 g(8.5 mmol) of the starting material ((E/Z)-diethyl2-(2-(3,5-diiodophenyl)hydrazinyl)hex-2-enedioate) dissolved in aminimum amount of dry toluene (ca. 15 ml) was added and the mixture wasrefluxed for 5 h. Then it was allowed to cool down to rt, and toluenewas removed under reduced pressure and the residue was dissolved indichloromethane and washed with water. The organic layer was dried overmagnesium sulfate, filtered, and evaporated to dryness. The residue waspurified by silica gel column chromatography using 20% of ethylacetate/cyclohexane as eluent to yield a light-beige-colored solid in72%. ¹H-NMR (DMSO-d₆) δ 1.17, 1.34 (each t, 3H, ³J=7.1 Hz, CH₃); 2.55(m, 2H, 2′-H); 3.53 (m, 2H, 1′-H); 4.06, 4.34 (each q, 2H, ⁴J=7.1 Hz,CH₂); 7.80 (d, 2H, J=1.7 Hz, 5-H, 7-H), 11.91 (s, 1H, NH). ¹³C-NMR(DMSO-d₆) δ 14.2 (2CH₃, each C═OOCH₂CH₃); 19.1 (C-2′); 36.1 (C-1′);59.9, 60.9 (2CH₂, each C═OOCH₂CH₃); 88.3 (C-4); 90.0 (C-6); 121.5 (C-7);122.1 (C-3); 125.4 (C-2); 126.0 (C-3a); 137.9 (C-7a); 138.4 (C-5); 161.1(2-CO₂Et); 171.9 (2-CO₂Et). LC-MS (m/z): 542 [M]⁺, 559 [M+NH4⁺]⁺, 540[M]⁻. purity (LC-SM): 98%.

(vi) 3-(2-Carboxyethyl)-4,6-diiodo-1H-indole-2-carboxylic acid

Ethyl 4,6-diiodo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate (1.71g, 5 mmol) was dissolved in 15 ml tetrahydrofurane (THF) with stirringat rt. Then a solution of 0.63 g of lithium hydroxide trihydrate (3equiv.) in 15 ml water was added and the resulting mixture was let tostir at rt for 24 h. After completion of the reaction THF was removedunder reduced pressure and the pH was adjusted to 4-5, and the productwas extracted with diethyl ether (3×15 ml). The organic layers weredried over magnesium sulfate, filtered, and evaporated to dryness toyield a light-beige-colored solid in 95-100% yield. mp. 280-282° C.¹H-NMR (DMSO-d₆) δ 2.48 (m, 2H, 2′-H); 3.49 (m, 2H, 1′-H); 7.78 (dd, 2H,J=1.7 Hz, 5-H, 7-H); 11.80 (s, 1H, NH); 12.68 (b, 2H, 2CO₂H). ¹³C-NMR(DMSO-d₆) δ 18.9 (C-2′); 36.4 (C-1′), 88.3 (C-4); 89.5 (C-6); 121.4(C-7); 122.0 (C-3); 126.2 (C-2); 126.4 (C-3a); 137.8 (C-7a); 138.1 (C7);162.7 (2′-CO₂H); 173.58 (2-CO₂H). LC-MS (m/z): 503 [M+NH4⁺]⁺, 486 [M]⁺,484 [M]⁻. Purity (LC-MS): 98%.

(j) 3-(2-Carboxyethyl)-4,6-dichloro-5-fluoro-1H-indole-2-carboxylic acid(i) 3,5-Dichloro-4-fluorobenzenediazonium salt (Mixture I)

To a well stirred suspension of 3,5-dichloro-4-fluoroaniline (0.90 g, 5mmol) in 8.3 ml HCl (5 M) at 0° C. was dropwise added a solution ofsodium nitrite 0.69 g (10 mmol, 2 equiv) in 4 ml water, previouslycooled to 0° C. The addition of sodium nitrite solution was slow, inorder to keep the temperature of the mixture below 8° C. The resultingorange-red mixture was stirred at 0° C. for further 20 min.

(ii) 2-(Ethoxycarbonyl)cyclopentanone (mixture II)

2-(Ethoxycarbonyl)cyclopentanone (1.26 ml, 0.67 g, 7.5 mmol) wasdissolved in ethanol (4.2 ml) and cooled to 0° C. Then, a potassiumhydroxide solution (2.52 g, 45 mmol, 9 equiv.) in water (5 ml)previously cooled to 0° C. was added dropwise within ca. 30 min in orderto keep the temperature below 8° C. The mixture turned milky-white andthe final mixture was stirred at 0° C. for further 30 min.

(iii)(E/Z)-5-(2-(3,5-Dichloro-5-fluorophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoicacid

Ice (50 g) was added to mixture II with stirring at 0° C., followed bythe addition of mixture I, and stirring continued for 1 h at 40° C. Thecombined mixtures were then let to cool to rt and the pH wassubsequently adjusted to 4-5 by adding 1 M HCl. The desired product wasextracted with diethyl ether (3×50 ml). The organic layer was collected,dried over magnesium sulfate, filtered, and the filtrate was evaporatedto dryness yielding gummy material (95-99%). This material was usedwithout further purification for the next step.

(iv) (E/Z)-Diethyl2-(2-(3,5-dichloro-4-fluorophenyl)hydrazinyl)hex-2-enedioate

(E/Z)-5-(2-(3,5-Dichloro-4-fluorophenyl)hydrazinyl)-6-ethoxy-6-oxohex-4-enoicacid (1.83 g, 5 mmol) was dissolved in absolute ethanol (100 ml)followed by the addition of conc. sulfuric acid (1.35 ml, 25.3 mmol, 5.1equiv.). The mixture was then allowed to reflux for 1 h at 100° C. Thenthe ethanol was evaporated and the residue was treated with 100 ml ofice-water. The aqueous solution was extracted with dichloromethane (3×50ml); the organic layer was dried over magnesium sulfate, filtered andconcentrated. The residue was purified by silica gel columnchromatography using 20% of ethyl acetate in cyclohexane yielding awhite solid in 85% yield.

(v) Ethyl4,6-dichloro-4-fluoro-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate

A mixture of p-toluenesulfonic acid (1.22 g, 6.4 mmol, 1.5 equiv.) and60 ml of dry toluene was refluxed for 1 h at 140° C.; water wascontinuously removed by means of a Dean-Stark trap. Subsequently, 1.67 g(4.25 mmol) of the starting material ((E/Z)-diethyl2-(2-(3,5-dibromophenyl)hydrazinyl)hex-2-enedioate) dissolved in aminimum amount of dry toluene (ca. 15 ml) was added and the mixture wasrefluxed for 2 h. Then it was allowed to cool down to rt, and toluenewas removed under reduced pressure and the residue was dissolved indichloromethane and washed with water. The organic layer was dried overmagnesium sulfate, filtered, and evaporated to dryness. The residue waspurified by silica gel column chromatography using 20% of ethylacetate/cyclohexane as eluent to yield a light-beige-colored solid in84%. ¹H-NMR (DMSO-d₆) δ 1.14, 1.34 (each t, 3H, ³J=7.1 Hz, CH₃); 2.54(m, 2H, 2′-H); 3.48 (m, 2H, 1′-H); 4.04, 4.35 (each q, 2H, ⁴J=7.1 Hz,CH₂); 7.50 (d, 1H, ²J=6 Hz, 7-H); 12.08 (s, 1H, NH). ¹³C-NMR (DMSO-d₆) δ14.1 (2CH₃, each C═OOCH₂CH₃); 20.1 (C-2′); 35.9 (C-1′); 59.9, 61.0(2CH₂, each C═OOCH₂CH₃); 112.6 (C-7); 113.0, 113.2, 117.9, 188.1, 121.4,121.5 (C-5); 122.1 (C-4); 126.8 (C-6); 132.7 (C-3); 147.3 (C-3a); 149.1(C-2); 160.9 (2′-CO₂Et), 171.9 (2-CO₂Et). LC-MS (m/z): 377 [M-NH4⁺]⁺,360 [M]⁺, 358 [M]⁻. Purity (LC-MS): 96%.

(vi) 3-(2-Carboxyethyl)-4,6-dichloro-4-fluoro-1H-indole-2-carboxylicacid

Ethyl4,6-dichloro-4-fluoro-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate(1.13 g, 3 mmol) was dissolved in 15 ml tetrahydrofurane (THF) withstirring at rt. Then a solution of 0.38 g of lithium hydroxidetrihydrate (3 equiv.) in 15 ml water was added and the resulting mixturewas let to stir at rt for 24 h. After completion of the reaction THF wasremoved under reduced pressure and the pH was adjusted to 4-5, and theproduct was extracted with diethyl ether (3×15 ml). The organic layerswere dried over magnesium sulfate, filtered, and evaporated to drynessto yield a light-beige-colored solid in 95-100% yield. mp. 292-294° C.¹H-NMR (DMSO-d₆) δ 2.48 (m, 2H, 2′-H); 3.48 (m, 2H, 1′-H); 7.49 (dd, 1H,²J=6 Hz, 7-H); 11.98 (s, 1H, NH); 12.72 (b, 2H, 2CO₂H). ¹³C-NMR(DMSO-d₆) δ 20.1 (C-2′); 36.1 (C-1′); 112.5 (C-7); 113.0, 113.2, 117.6,117.7, 121.3, 121.4 (C-5); 122.3 (C-4); 127.7 (C-6); 132.5 (C-3); 147.2(C-3a); 149.0 (C-2); 162.5 (2′-CO₂H), 173.6 (2-CO₂H). LC-MS (m/z): 337[M-NH4⁺]⁺, 320 [M]⁺, 318 [M]⁻. Purity (LC-MS): 98.6%.

(I) 3-(2-Carboxyethyl)-4,6-diphenyl-1H-indole-2-carboxylic acid (i)Ethyl 4,6-diphenyl-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate

Ethyl 4,6-dibromo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate(0.244 g, 0.5 mmol), phenylboronic acid (0.147 g, 1.2 mmol, 2.4 equiv.)and potassium phosphate trihydrate (0.400 g, 1.5 mmol, 3.0 equiv.) weremixed together in a 10 ml microwave vial. The vial was purged with Argonand trans-dichlorobis(triphenylphosphine)-palladium (II) (0.021, 0.03mmol, 6 mol %) and 5 ml of dry dioxane were added to the mixture. Themicrowave vial was capped and irradiated at 100 watt, 150° C., underpressure up to 10 bars. Then it was allowed to cool down to rt, andtoluene was removed under reduced pressure and the residue was dissolvedin dichloromethane and washed with water. The organic layer was driedover magnesium sulfate, filtered, and evaporated to dryness. The residuewas purified by silica gel column chromatography using 20% of ethylacetate/cyclohexane as eluent to yield a light-beige-colored solid in60-70%. ¹H-NMR (DMSO-d₆) δ 1.09, 1.32 (each t, 3H, ³J=7.1 Hz, CH₃); 2.11(m, 2H, 2′-H); 2.84 (m, 2H, 1′-H); 3.89, 4.33 (each q, 2H, ⁴J=7.1 Hz,CH₂); 7.12 (d, 1H, ²J=1.6 Hz, 7-H); 7.35 (t, 1H, ³J=7.4 Hz, Ph-ring);7.43 (m, 7H, Ph-ring); 7.67 (d, 1H, ²J=0.9 Hz, 5-H); 7.68 (d, 1H, ²J=1.6Hz, Phenyl-ring); 11.84 (s, 1H, NH). ¹³C-NMR (DMSO-d₆) δ 14.2, 14.3(2CH₃, each C═OOCH₂CH₃); 20.5 (C-2′); 34.8 (C-1′); 59.5, 60.5 (2CH₂,each C═OOCH₂CH₃); 109.6 (C-5); 121.1 (C-7); 121.8 (C-3); 123.6 (C-2);124.7 (C-3a); 127.0 (C-2″, C-6″); 127.4 (C-4″′); 127.5 (C-4″); 128.0(C-3″′, C-5″′); 129.0 (C-3″, C-5″); 129.1 (C-2″′, C-6″′); 136.7 (C-7a);137.4 (C-4), 137.6 (C-6); 140.7 (C-1″); 140.8 (C-1″′); 161.5 (2′-CO₂Et);171.7 (2-CO₂Et). LC-MS (m/z): 442 [M]⁺, 440 [M]⁻. Purity (LC-MS): 96%.

(ii) 3-(2-Carboxyethyl)-4,6-diphenyl-1H-indole-2-carboxylic acid

Ethyl 4,6-diphenyl-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate(4.42 g, 10 mmol) was dissolved in 25 ml tetrahydrofurane (THF) withstirring at rt. Then a solution of 1.26 g of lithium hydroxidetrihydrate (3 equiv.) in 25 ml water was added and the resulting mixturewas let to stir at rt for 24 h. After completion of the reaction THF wasremoved under reduced pressure and the pH was adjusted to 4-5, and theproduct was extracted with diethyl ether (3×25 ml). The organic layerswere dried over magnesium sulfate, filtered, and evaporated to drynessto yield a light-beige-colored solid in 95-100% yield. mp. 243-245° C.¹H-NMR (DMSO-d₆) δ 2.07 (m, 2H, 2′-H); 2.83 (m, 2H, 1′-H); 7.09 (d, 1H,²J=1.6 Hz, 7-H); 7.34 (t, 1H, ³J=7.4 Hz, ph-ring), 7.44 (m, 7H,Ph-ring), 7.66 (d, 2H, ²J=0.9 Hz, 5-H); 7.68 (d, 1H, 1.6 Hz, Ph-ring);11.72 (s, 1H, NH); 12.33 (b, 2H, 2CO₂H). ¹³C-NMR (DMSO-d₆) δ 20.3(C-2′); 34.9 (C-1′); 109.5 (C-4); 120.9 (C-6); 121.7 (C-3); 123.8 (C-2);125.5 (C-3a); 127.0 (C-2″, C-6″); 127.3 (C-4″′); 127.5 (C-4″); 128.0(C-3″′, C-5″′); 129.0 (C-3″, C-5″); 129.1 (C-2″′, C-6″′); 135.4 (C-7a);137.3 (C-4), 137.4 (C-6); 140.8 (C-1″); 141.0 (C-1″′); 163.1 (2′-CO₂H);173.4 (2-CO₂H). LC-MS (m/z): 386 [M-NH₄ ⁺]⁺, 366 [M]⁺, 384 [M]⁻. Purity(LC-MS): 95%.

(k) 3-(2-Carboxyethyl)-6-phenyl-1H-indole-2-carboxylic acid (i) Ethyl6-phenyl-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate

Ethyl 6-bromo-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate (0.185 g,0.500 mmol), phenylboronic acid (0.76 g, 0.625 mmol, 1.2 equiv.) andpotassium phosphate trihydrate (0.200 g, 0.750 mmol, 1.5 equiv.) weremixed together in a 10 ml microwave vial. The vial was purged with Argonand trans-dichlorobis(triphenylphosphine)-palladium (II) (0.0105 g,0.015 mmol, 3 mol %) and 5 ml of dry dioxane were added to the mixture.The microwave vial was capped and irradiated at 100 watt, 150° C., underpressure up to 10 bars. Then it was allowed to cool down to rt, andtoluene was removed under reduced pressure and the residue was dissolvedin dichloromethane and washed with water. The organic layer was driedover magnesium sulfate, filtered, and evaporated to dryness. The residuewas purified by silica gel column chromatography using 20% of ethylacetate/cyclohexane as eluent to yield a light-beige-colored solid in70-80%. ¹H-NMR (DMSO-d₆) δ 1.11, 1.35 (each t, 3H, ³J=7.1 Hz, CH₃); 2.60(m, 2H, 2′-H); 3.31 (m, 2H, 1′-H); 3.99, 4.35 (each q, 2H, ⁴J=7.1 Hz,CH₂); 7.35 (m, 2H, 4-H, 5-H); 7.46 (m, 2H, 2″-H, 6″-H); 7.62 (dd, 1H,4″-H); 7.65 (m, 2H, 3″-H, 5″-H); 7.75 (dd, 1H, 7-H); 11.63 (s, 1H, NH).¹³C-NMR (DMSO-d₆) δ 14.9, 15.1 (2CH₃, each C═OOCH₂CH₃); 20.90 (C-2′);35.9 (C-1′); 60.7, 61.2 (2CH₂, each C═OOCH₂CH₃); 111.0 (C-7); 120.1(C-4); 121.8 (C-3); 122.3 (C-3a), 124.8 (C-5) 127.2 (C-2″, C-6″); 127.7(C-4″); 128.0 (C-3″, C-5″); 129.8 (C-2); 137.7 (C-7a); 138.2 (C-6);141.9 (C-1″); 162.3 (2′-CO₂Et); 173.1 (2-CO₂Et). LC-MS (m/z): 383 [M-NH₄⁺]⁺, 366 [M]⁺, 364 [M]⁻. Purity LC-MS: 96%.

(ii) 3-(2-Carboxyethyl)-6-phenyl-1H-indole-2-carboxylic acid

Ethyl 6-phenyl-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-carboxylate (3.65 g,10 mmol) was dissolved in 25 ml tetrahydrofurane (THF) with stirring atrt. Then a solution of 1.26 g of lithium hydroxide trihydrate (3 equiv.)in 25 ml water was added and the resulting mixture was let to stir at rtfor 24 h. After completion of the reaction THF was removed under reducedpressure and the pH was adjusted to 4-5, and the product was extractedwith diethyl ether (3×25 ml). The organic layers were dried overmagnesium sulfate, filtered, and evaporated to dryness to yield alight-beige-colored solid in 95-100% yield. mp. 238-239° C. ¹H-NMR(DMSO-d₆) δ 2.54 (m, 2H, 2′-H); 3.28 (m, 2H, 1′-H); 7.35 (m, 2H, 4-H,5-H); 7.46 (m, 2H, 2″-H, 6″-H); 7.60 (dd, 1H, 4″-H); 7.63 (m, 2H, 3″-H,5″-H); 7.75 (dd, 1H, 7-H); 11.55 (s, 1H, NH); 12.50 (b, 2H, 2CO₂H).¹³C-NMR (DMSO-d₆) δ 20.1 (C2′); 35.3 (C1′); 110.2 (C-7); 119.2 (C-5),121.1 (C-4); 121.4 (C-3); 124.8 (C-3a); 126.6 (C-2); 127.0 (C-3″, C-5″);127.2 (C-4″); 129.1 (C-3″, C-6″); 136.8 (C-7a); 137.2 (C-6); 141.3(C-1″); 163.1 (2′-CO₂H); 174.1 (2-CO₂H). LC-MS (m/z): 310 [M]⁺, 308[M]⁻. Purity (LC-MS): 98%.

The compounds listed in Table 2 can be produced using the methods andmanufacturing routes similar to and based on the manufacturing schemesdisclosed in the experimental section hereinbefore, which can be adaptedby one skilled in the art, accordingly.

The invention claimed is:
 1. A compound selected from3-(2-carboxyethyl)-4,6-dibromo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dichloro-(1-carboxyethyl)-indole-2-carboxylicacid, 3-(2-carboxyethyl)-6-bromo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-iodo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-diiodo-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-diphenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-phenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6,7-dichloro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-chloro-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-bromo-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-benzyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-4,6-dihydroxy-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-(4-fluorophenyl)-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-furanyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-thienyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-7-fluoro-6-phenyl-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-(4-fluorophenyl)-7-fluoro-1H-indole-2-carboxylicacid, 3-(2-carboxyethyl)-6-furanyl-7-fluoro-1H-indole-2-carboxylic acid,3-(2-carboxyethyl)-6-thienyl-7-fluoro-1H-indole-2-carboxylic acid, andsalts of any of the foregoing.